Nonspecific acid phosphatase from Schizosaccharomyces pombe. Purification and physical chemical properties. Repressible nonspecific acid phosphatase from Schizosaccharomyces pombe was purified to apparent homogeneity, as ascertained from ultracentrifugal, electrophoretic, and chromatographic data. The native protein has a molecular weight of 383,000 as determined by sucrose density gradient centrifugation and 381,000 as determined by gel filtration. The native protein can be dissociated in the presence of 8 M urea-1% sodium dodecyl sulfate into sub-units possessing an approximate molecular weight of 104,000. Neutral sugars account for about 66% of the total molecular weight and contribute to the high solubility and some of the other physical properties of this enzyme. Purified enzyme preparations have a Km for 4-nitrophenyl phosphate of 0.17 mM and a broad substrate specificity, but do not show diesterase activity. Phosphate and sulfate are competitive inhibitors. The enzyme is inactivated at neutral and alkaline pH and at relatively low temperatures. Mannose and galactose was found as the main components of the carbohydrate moiety; glucosamine was present in lower amounts. The amino acid analysis revealed a high content of aspartate, threonine, and serine; no sulfhydryl group could be detected. Pi is released in stoichiometric amount (1 mol per enzyme monomer) on protein digestion.


Comparison of glucocorticoid-binding proteins in normal and neoplastic mammary tissues of the rat. Kinetic and molecular properties of components binding [3H]triamcinolone acetonide were studied using 105,000g supernatants of lactating mammary gland, R3230AC, and dimethylbenz[a]anthracene (DMBA) induced mammary tumors of the rat. Using a dextran-coated charcoal adsorption procedure, the relationship between specific glucocorticoid binding and protein concentration was linear in the range of 0.5-4.0 mg/reaction. These cytoplasmic macromolecules bound [3H]triamcinolone acetonide with limited capacity (50-400 fmol/mg of cytosol protein) and high affinity, Kd approximately 10(-8)-10(-9) M. Optimal binding was obtained when homogenizations were made in Tris buffers, at pH 7.4, containing monothioglycerol. Time course of association of [3H]triamcinolone acetonide and its binding sites showed maximal binding by 6-8 hr at 3 degrees which remained unchanged up to 24 hr. The rate constant of association at 3 degrees was in the range of 2-4 x 10(5) M-1 min-1. The rate constant of dissociation of bound [3H]triamcinolone acetonide could not be calculated accurately since the reaction was essentially irreversible for 5 hr at 3 degrees. Estimation of the half-life of the steroid-binding protein complexes from the Kd and the rate constant for association gave a value of 11-12 hr. From ligand specificity studies, the glucocorticoids, triamcinolone acetonide, corticosterone, cortisol, and dexamethasone competed well for [3H]triamcinolone acetonide binding sites. Progesterone, aldosterone, and the anti-glucocorticoid, cortexolone, were also good competitors while androgens and estrogens were weak inhibitors of binding. The binding compenents sedimented at 7-8 S in sucrose gradients of low ionic strength and dissociated into lower molecular weight components sedimenting at 4-5S in high ionic strength gradients. Studies in vivo using animals bearing the DMBA-induced tumor demonstrated that [3H]triamcinolone acetonide binding complexes were present in cytoplasmic and nuclear compartments. Sedimentation coefficients of the cytoplasmic and nuclear forms of these receptors labeled in vivo were 7-8S and 4-5S, respectively. These studies suggest that the molecular and kinetic binding properties of glucocorticoid receptors in neoplastic mammary tissues are similar to those of the normal mammary gland.


Heavy metal-nucleoside interactions. Binding of methylmercury(II) to inosine and catalysis of the isotopic exchange of the C-8 hydrogen studied by 1-H nuclear magnetic resonance and raman difference spectrophotometry. Raman difference spectrophotometry reveals that CH3HgII binds quantitatively to N(1) of inosine at pH 8, substituting for the proton. When N(1) is saturated, binding occurs at a second site. Measurements of the 1-H nuclear magnetic resonance spectra of both inosine and of CH3Hg-II are in agreement with the N(1) binding and indicate that the second site for mercuriation is N(7). This second binding reaction is observed to increase the rate of exchange of the C(8) hydrogen with solvent, consistent with results observed for alkylation at N(7). Coordination of the electrophilic CH3Hg-II to N(7) increases the acidity of H(8), facilitating OHminus--catalyzed proton abstraction and reprotonation by themedium. For comparison, the reaction of CH3Hg-II with [8-2-H]inosine has been studied. Displacement of the N(1) hydrogen upon mercuriation of inosine causes a significant electron delocalization into the ring, increasing the basicity of N(7), and accounting for the synergic effect in metal binding observed originally by Simpson. In contrast, 1-methylinosine interacts only slightly with CH3Hg-II at pH 8. Coordination appears to be at N(7), since H(8) again is observed to exchange rapidly with solvent protons. In acidic solution, pH less than 2, binding to inosine is almost quantitative and exclusively to N(7). The behavior of CH3Hg-II is compared with that of Pt(II) and with Ni(II), Co(II), AND Zn(II). A brief comparison is made among ultraviolet absorption spectrophotometry, nuclear magnetic resonance (NMR), and Raman difference spectrophotometry for studying reactions of nucleosides and nucleotides.


Ionic and nucleotide requirements for microtubule polymerization in vitro. The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.


Reactions of fluorescent probes with normal and chemically modified myelin. The fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind to highly purified myelin membranes obtained from bovine brain white matter. Binding of the dyes was markedly increased by environmental conditions which reduce the negative surface potential of the membrane, i.e., cations (La-3+ is greater than Ca-2+ is greater than Na-+,K-+), H-+, local anesthetics, and the antibiotic polymyxin B. Chemical alteration of accessible membrane charged groups affected dye binding in a manner consistent with the hypothesis that such binding is primarily dependent upon the membrane surface potential. Thus, binding was increased by blocking of carboxyl groups via carbodiimide activation and subsequent coupling with neutral amino acid esters, and even more so with a basic amino acid ester (e.g., arginine methyl ester). Dye binding was reduced by succinylation of amino groups, and by hydrolysis of choline and ethanolamine head groups of phospho- and sphingolipids by phospholipase C. Phospholipase C treatment of myelin, or sphingomyelin vesicles, reduced or abolished the augmentation of ANS and TNS binding due to cations, local anesthetics, or polymyxin B. Energy transfer from myelin tryptophan residues to bound ANS occurs, but with low efficiency. Oxidation of membrane tryptophan residues with N-bromosuccinimide, or alkylation with 2-hydroxy (or methoxy)-5-nitrobenzyl bromide, markedly reduced intrinsic membrane fluorescence and energy transfer to bound ANS, but did not significantly affect dye binding or the quantum yield of ANS fluorescence when excitation was at 380nm. Proteolytic digestion removed 6-30% of myelin protein, depending upon the enzyme used, but had no effect on fluorescent dye binding. It is concluded that the binding of the anionic fluorescent probes ANS and TNS to myelin is primarily a function of the membrane surface charge density and net surface potential, as is the case with other biological membranes. Conclusions about the degree of dye binding to membrane lipids or membrane proteins cannot be drawn unless additional studies are carried out on isolated water soluble membrane proteins.


Circular dichroism and gel filtration behavior of subtilisin enzymes in concentrated solutions of guanidine hydrochloride. The circular dichroism of diisopropylphosphorylsubtilisins Novo and Carlsberg in both the near- and farultraviolet spectral regions is unaltered by concentrations of guanidine hydrochloride as high as 4 M at neutral pH. At concentrations of guanidine hydrochloride greater than 4 M slow irreversible time-dependent changes, apparently obeying second-order kinetics, are evident in both the near- and far-ultraviolet circular dichroism of these enzymes. Gel filtration studies of inactivated subtilisin enzymes reveal the circular dichroism changes to be accompained by the appearance of aggregated protein material. The changes in circular dichroism and the production of associated subtilisin species are sensitive to protein concentration, denaturant concentrations, and pH. The circular dichroism of active subtilisins Novo and Carlsberg in guanidine hydrochloride exhibits irreversible changes similar to those observed for the inactivated subtilisins. Aggregated protein material is also formed initially in the presence of guanidine hydrochloride, but is rapidly autolyzed to low molecular weight fragments.


H nuclear magnetic resonance study of restricted internal rotation of N-6,N-6-dimethyladenine in aqueous solution. Kinetics of internal rotation about the C(6)-N(6) bond of N-6,N-6-dimethyladenine (M2-6A) was investigated by -1H nuclear magnetic resonance line-shape analysis of the methyl resonances (220 MHz). Rates of rotation were determined for M2-6A deuterated at N(1) and for neutral M2-6A. Activation parameters for monodeuterated M2-6A at 22 degrees are Ea = 13.8kcal/mol, log A = 12.6, incrementG++=14.9 kcal/mol, incrementH++ = 13.1 kcal/mol, incrementS++ = minus 5.8 eu; for neutral M2-6A: Ea = 15.5 kcal/mol, log A = 14.9, incrementG++ = 12.6 kcal/mol, incrementH++ = 14.9 kcal/mol, incrementS++ =7.8 eu. Vertical stacking of bases interferes with internal rotation of the dimethylamino group.


Intermolecular complexes between N-methyl-1,4-dihydronicotinamide and flavines. The influence of steric and electronic factors on complex formation and the rate of flavine-dependent dihydronicotinamide dehydrogenation. The reaction of N-methyldihydronicotinamide (NMNH) with flavine analogs saturates at high dihydronicotinamide concentrations. Complex formation between the reactants depends mainly on steric but not on electronic factors. Thus flavine analogs that differ up to 243 mV in their oxidation-reduction potential vary only between 0.09 and 0.17 M in Kd. When the flavine plane becomes blocked by bulky substituents, however, complex stability decreases by more than an order of magnitude. NMNH-flavine complexes show long wave optical absorption. The energy of the long wave transition decreases with increasing oxidation-reduction potential of the flavine as expected for charge transfer complexes. The first-order rate constants of flavine-dependent dihydronicotinamide dehydrogenation increase with increasing oxidation-reduction potential of the flavine but they are almost independent of Kd. The reaction is not subject to general acid-base catalysis. Thus flavine-dependent dihydronicotinamide dehydrogenation may be interpreted to proceed via a charge transfer complex between oxidized flavine and reduced nicotinamide. In the rate-limiting conversion of the charge transfer complex into products hydrogen is transferred directly, the rate being governed by the difference in oxidation-reduction potential between flavine and dihydronicotinamide. An alternative mechanism where the observed charge transfer complex is not on the reaction pathway appears to be improbable but cannot be eliminated.


Roles of zinc ion and reduced coenzyme in horse liver alcohol dehydrogenase catalysis. The mechanism of aldehyde activation. 1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step.


Nitrosylhemoglobin Wood: effects of inositol hexaphosphate on thiol reactivity and electron paramagnetic resonance spectrum. Properties of Hb Wood (beta-97(FG4)His leads to Leu), a high oxygen affinity hemoglobin with reduced hemeheme interaction, were examined in its nitric oxide liganded form. The reactivity of the beta-93 thiol groups and the electron paramagnetic resonance (EPR) spectrum were examined to determine what effect the amino acid substitution, which occurs at the alpha1beta2 interface, would have on inositol hexaphosphate induced transition of this form of the tetramer. Binding of inositol hexaphosphate (IHP) in a 1:1 stoichiometry was demonstrated. In spite of apparently normal interaction with IHP, there was little or no change in the reactivity of the beta-93 thiol groups and in the electron paramagnetic resonance (EPR) spectrum as contrasted with the marked changes characteristic of normal hemoglobin (HbA). In contrast with NO-HbA, there was also no development of the EPR hyperfine structure in NO-Hb Wood with increased protonation of the protein at pH below 7.0. Taken together with the observations of Henry and Banerjee ((1973), J. Mol. Biol. 73, 469) on the development of NO-Hb EPR hyperfine structure and of Perutz et al. (1974a), Biochemistry 13, 2174) on changes in thiol reactivity with the R leads to T transition, the results suggest that IHP or H+ cannot switch NO-Hb Wood to the T conformation. Since the atomic structures of met- and deoxyhemoglobin offer no indication that His-97 plays any special part in the allosteric mechanism (M. E. Perutz, personal communication), it appears that the replacement of His-97 by Leu reduces the stability of the T structure relative to that of R.


Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique. Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].


An electrophoretic study of reversible protein denaturation: chymotrypsinogen at high pressures. When reversible denaturation of chymotrypsinogen is produced at elevated hydrostatic pressures, conformational relaxation can occur quite slowly, allowing electrophoretic separation of the principal states from the equilibrium mixture. In this work we report experimental concentration distribution patterns obtained at pH 2.03 at a temperature of 20.5 degrees and find them to be reasonably consistent with the behavior that is expected from a simple two-state isomerism. However, the results do not at all rule out the existence of low levels of intermediate states.


Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (Kunitz). Solvent accessibility in the folded conformation. The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, THE CONTRIBUTION OF MAJOR, REVERSIBLE PROTEIN UNFOLDING To the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but congruent to 8 protons can be observed to exchange with E*app congruent to 27 kcal/mol. This implies that all but congruent to 8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30 degrees, approximating STI structure by a sphere of radius = 18 A. These calculations indicate an average of congruent to 4 water molecules in the shell from 13 to 16 A. from the center of the molecule, while less than 1 water molecule is indicated in the innermost 13 A. These calculations also suggest that there are congruent to 190 water molecules associated with the outermost 1.5-2 A of the sphere. While these values are consistent with a hydrophobic region in the central protein matrix, they indicate more solvent accessibility in the outer 1/3 of the molecule than the static accessibility estimates made from X-ray coordinates. Our results suggest that any protein movements or fluctuations responsible for solvent accessibility in proton exchange processes are localized in the outer regions of the globular structure.


Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor-trypsin complex. The hydrogen exchange kinetics of the complex of trypsin-soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins.


High-resolution proton nuclear magnetic resonance studies of sickle cell hemoglobin. High-resoluiton proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate sickle cell hemoglobin. The hyperfine shifted, the ring-current shifted, and the exchangeable proton resonances suggest that the heme environment and the subunit interfaces of the sickle cell hemoglobin molecule are normal. These results suggest that the low oxygen affinity in sickle cell blood is not due to conformational alterations in the heme environment or the subunit interfaces. The C-2 proton resonances of certain histidyl residues can serve as structural probes for the surface conformation of the hemoglobin molecule. Several sharp resonances in sickle cell hemoglobin are shifted upfield from their positions in normal adult hemoglobin. These upfield shifts, which are observed in both oxy and deoxy forms of the molecule under various experimental conditions, suggest that some of the surface residues of sickle cell hemoglobin are altered and they may be in a more hydrophobic environment as compared with that of normal human adult hemoglobin. These differences in surface conformation are pH and ionic strength specific. In particular, upon the addition of organic phosphates to normal and sickle cell hemoglobin samples, the differences in their aromatic proton resonances diminish. These changes in the surface conformation may, in part, be responsible for the abnormal properties of sickle cell hemoglobin.


A kinetic study of the reaction between cytochrome c peroxidase and hydrogen peroxide. Dependence on pH and ionic strength. The rate of the reaction between cytochrome c peroxidase and hydrogen peroxide was investigated using the stopped-flow technique. The apparent bimolecular rate constant was determined between pH 3.3 and pH 11 as a function of ionic strength. The pH dependence of the apparent bimolecular rate constant can be explained by assuming that two ionizable groups on the enzyme strongly influence the rate of the reaction. At 0.1 M ionic strength, a group with a pKa of 5.5 must be unprotonated and a group with a pKa of 9.8 must be protonated for the enzyme to react rapidly with hydrogen peroxide. The apparent acid dissociation constants depend upon the ionic strength. The true bimolecular rate constant has a value of (4.5 +/- 0.3) X 10(7) M-1 sec-1 and is independent of ionic strength.


Characterization and properties of Pholas luciferase as a metalloglycoprotein. The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.


A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase. The binding of NADP+ to dihydrofolate reductase (EC 1.5.1.3) in the presence and absence of substrate analogs has been studied using 1H and 13C nuclear magnetic resonance (NMR). NADP+ binds strongly to the enzyme alone and in the presence of folate, aminopterin, and methotrexate with a stoichiometry of 1 mol of NADP+/mol of enzyme. In the 13C spectra of the binary and ternary complexes, separate signals were observed for the carboxamide carbon of free and bound [13CO]NADP+ (enriched 90% in 13C). The 13C signal of the NADP+-reductase complex is much broader than that in the ternary complex with methotrexate because of exchange line broadening on the binary complex signal. From the difference in line widths (17.5 +/- 3.0 Hz) an estimate of the dissociation rate constant of the binary complex has been obtained (55 +/- 10 sec-1). The dissociation rate of the NADP+-reductase complex is not the rate-limiting step in the overall reaction. In the various complexes studied large 13C chemical shifts were measured for bound [13CO]NADP+ relative to free NADP+ (upfield shifts of 1.6-4.3 ppm). The most likely origin of the bound shifts lies in the effects on the shieldings of electric fields from nearby charged groups. For the NADP+-reductase-folate system two 13C signals from bound NADP+ are observed indicating the presence of more than one form of the ternary complex. The IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments. Substantial changes in the IH spectrum of the protein were observed in the different complexes and these are distinct from those seen in the presence of NADPH.


Preparation and properties of three specific active derivatives of ribonuclease A obtained by methylation of methionine residues in 8 M urea. In 8 M urea at low pH, CH3I reacts specifically with the four methionine residues of ribonuclease A, and all four residues react at the same rate. Uon removal of the denaturant, only unmodified ribonuclease and 3 of the 15 possible derivatives modified on methionine refold to regenerate activity. All the enzymatic activity is recored after chromatography on IRC-50 and the four active proteins separate from each other and from the 12 inactive derivatives, which are not eluted from the resin under the conditions used. By the use of 14CH3I, performic acid oxidation, chymotryptic digestion, and separation of the resulting peptides by ion exchange, the active species were determined to be unmodified ribonuclease, CH3Met-29-RNase, CH3Met-79-RNase, and CH3Met-29, CH3Met-79-RNase. these proteins have melting temperatures of 63, 58, 43, and 36 degrees, respectively, at pH 6.3-70. Methylation at methionine-29 or -79 has no effect on enzymatic activity. Conversely, methylation at methionine-13 or -30 prevents refolding to an active conformation at 25 degrees elution from IRC-50. These results are consistent with the positions of the four methionine residues in crystals of ribonuclease A and ribonuclease S as determined by X-ray diffraction.


Gamma-glutamyltransferase from azo dye induced hepatoma and fetal rat liver.  Similarities in their kinetic and immunological properties. Some properties of gamma-glutamyltransferase ((gamma-glutamyl)-peptide: amino-acid gamma-glutamyltransferase EC 2.3.2.2) from azo dye induced hepatoma and fetal rat liver were studied using kinetic and immunological criteria. There was no significant difference between the hepatoma enzyme and fetal rat liver enzyme in some of their catalytic properties. Antisera against the purified hepatoma enzyme also reacted to the fetal rat liver enzyme in the inhibition test and the precipitin reaction. A structural similarity between the hepatoma enzyme and fetal rat liver enzyme was observed and the acquirement of fetal characteristics in hepatoma was discussed.


Surface-bound aspartate aminotransferase on collagen films.  Compared properties with native enzyme. Aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) has been covalently bound to chemically activated collagen films. This enzyme had never previously been coupled to any other solid support. The coupling method, including acyl azide formation on the carrier, allowed coupling of many other enzymes. A systematic study of coupling conditions has been performed; influence of time of coupling and of concentration of coupling solution on the enzymatic activity retained on the film. Coupling solutions could be used for several successive couplings. To determine the yield of binding, N-[14C] ethylmaleimide-labelled enzyme was prepared fully active and bound to collagen films. After lyophilisation the film retained most of its activity when stored in buffer and the half-life of the enzymatic film was about ten months. pH Dependence and activation energy were about the same for soluble and coupled enzyme. Coupling protects against thermal denaturation and increases the stability of the enzyme; the enzymatic film could be used repeatedly. Kinetics were somewhat modified in the coupled enzyme as compared to the enzyme in solution. Glutamate appeared more available while oxaloacetate seemed to be limiting. These modifications might be due to the proteic support itself. The enzymatic films also revealed themselves as a good tool for industrial or clinical purposes as well as for studying the mechanism of enzyme action.


The reaction of tris (hydroxymethyl) aminomethane with calf intestinal alkaline phosphatase. The effect of tris (hydroxymethyl) aminomethane concentrations on the rate of calf intestinal alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate was studied, in the pH range 8-10, where no transphosphorylation reaction could be detected. Kinetic analysis of the results permitted description of the effect of Tris concentrations T on the rate of enzyme catalyzed hydrolysis (V) by the following equation: (see article). The rate-accelerating effect of Tris concentrations can be ascribed to two different mechanisms: At moderate Tris concentrations (0.01-0.20 M) the enzyme forms a reversible addition complex with a Tris molecule. This complex has an enhanced catalytic activity. We suggest that the binding of Tris to the enzyme could potentiate a second active site of the enzyme, due to its ionization effect upon an acidic group of the enzyme of pK = 8.9. The modest linear rate accelerating effect of Tris at high concentrations (0.20-0.60 M) could be ascribed to the change of the dielectric constant of the medium, the degree of solvation of the protein, or change in the tertiary structure of the enzyme.


Resolution, purification and characterization of the orthophosphate releasing activities from fracture callus calcifying cartilage. Callus calcifying cartilage alkaline phosphatase was resolved by DEAE-cellulose column chromatography into two distinct phsophatase activities. The phosphatase activity which was eluted first from the column, (phosphatase I), was active towards a variety of phosphate esters, sodium pyrophosphatase and several linear polyphosphates, while the second phosphatase activity , (phosphatase II), was active toward simple phosphate esters but not towards sodium pyrophosphate and linear oligo or polyphosphates. All the phosphate esters, sodium pyrophosphate and polyphosphates at higher concentrations were inhibitory for phosphatase I. The modulating effects of magnesium, calcium, zinc and other phosphatase modulators have been investigated. Both phosphatases from callus calcifying cartilage were found to be substrates of neuraminidase with sialic acid as the product. Besides the difference in their specificity, the phosphatases were found to be immunologically different and to have different molecular weights, strong indication that they are different enzymes.


The relationship between different forms of human alpha-mannosidase. The tissue distribution and some properties of human alpha-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) have been studied. The acidic forms of the enzyme were fairly stable, whereas the neutral forms easily lost enzymic activity. The acidic forms were sensitive to neuraminidase but the neutral forms were unaffected. The experiments indicate that the acidic components are closely related to each other, differing only in sialic acid content and possibly conformation. The neutral forms of the enzyme are probably quite different from the acidic forms both in structure and cellular function.


Purification and characterization of Aspergillus niger exo-1,4-glucosidase. A specific exo-1,4-glucosidase (1,4-alpha-D-glucan glucohydrooase, EC 3.2.1.3) from Aspergillus niger has been partially purified and subsequently characterized by biochemical, physico-chemical and optical methods. Molecular sieve chromatography yields an enzyme with maximal activity at pH 4.2-4.5 close to its isoelectric point. Reduction and carboxymethylation leads to complete loss of activity and O-acetylation of 3 of the 13 tyrosine residues results in loss of 20 % of the activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that the native enzyme consists of two major components of molecular weights 63 000 and 57 500, respectively. Small amounts of dissociated material of molecular weight 28 000 and 16 000 as well as aggregates of the order of 100 000 are also present to the extent of 2-5% of the total potein. Following reduction and carboxymethylation under forcing conditions, the bands around 60 000 diminish and the 28 000-30 000, 16 000 and aggregate bands are dominant...


Formation of a covalent intermediate between alpha-chymotryspin and the B-chain of insulin during enzyme-catalyzed hydrolysis. The reaction between alpha-chymotrypsin (EC 3.3.21.1) and the B-chain of bovine insulin was studied radiochemically, by using the 3 5S-labelled sulfo B-chain. After incubation at pH 8.0, interrupted by the addition of trichloroacetic acid, a radioactive product was isolated from the reaction mixture. The labelled product was eluted in parallel with the enzyme in gel chromatography, and its properties at different H+ concentrations indicated that chemically it was an ester, i.e. a covalent enzyme-substrate intermediate. No interaction between sulfo beta-chain and alpha-chymotrypsinogen or phenyl-methyl sulfonyl fluoride-inhibited alpha-chymotrypsin was obtained during identical conditions.


Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe. The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.


Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa. A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.


The effect of Mg2+ and chelating agents on intermediary steps of the reaction ofNa+,K+-activated ATPase. (1) It has been investigated how varying concentrations of free magnesium with and without EDTA influence the properties of the phospho-enzyme formed in the presence of sodium by the (Na+ plus K+)-activated enzyme system. (2) The phospho-enzyme formed in the presence of sodium and a high concentration of free magnesium has the same rate of (a) spontaneous dephosphorylation, (b) dephosphorylation after addition of potassium, and (c) dephosphorylation after addition of ADP, as a phospho-enzyme formed in the presence of sodium and a low concentration of magnesium. (3) With sodium and a given concentration of free magnesium, high or low, EDTA present during formation of the phospho-enzyme leads to a decrease in the rate of (a) spontaneous dephosphorylation, and (b) dephosphorylation after addition of potassium to the phospho-enzyme. (4) The rate of dephosphorylation after addition of ADP to phospho-enzyme formed without and with EDTA is the same. But as the rate of spontaneous dephosphorylation is lower with EDTA than without, ADP gives a higher increase in the rate of dephosphorylation of phospho-enzyme formed with EDTA than without. (5) The experiments thus show that the reported different sensitivity towards potassium and ADP of phospho-enzyme formed in the presence of a low and high concentration of free magnesium, respectively, is due to the EDTA used to decrease the free magnesium concentration and not to the decrease in the free magnesium as such.


Inhibition of horse muscle acylphosphatase by pyridoxal 5'-phosphate. It has been shown that horse muscle acylphosphatase is inhibited by pyridoxal 5'-phosphate and that the inhibition is pH dependent, reversible and competitive with respect to substrate binding. Spectral analysis on the EI complex demonstrates the presence of a Schiff base. Reduction of the pyridoxal 5'-phosphate-inhibited enzyme with sodium borohydride, followed by amino acid analysis, produces a diminution of the free lysine peak and the appearance of a new peak corresponding to epsilon-pyridoxyllysine. The results suggest that there is at least one NH2-lysyl residue of horse muscle acylphosphatase at or near the active site of the enzyme.


The oxidation of cytochrome c peroxidase by hydrogen peroxide. Characterization of products. H2O2 reacts with cytochrome c peroxidase in a variety of ways. The initial reaction produces cytochrome c peroxidase Compound I. If more than a 10-fold excess of H2O2 is added to the enzyme, a portion of the H2O2 will react with Compound I to produce molecular oxygen. The remainder oxidizes the heme group and various amino acid residues in the protein. If less than a 10-fold excess of H2O2 is added to the enzyme, essentially all the H2O2 is utilized by oxidation of amino acid residues in the protein. The oxidation of the amino acid residues by H2O2 substantially modifies the reactivity of cytochrome c peroxidase. The modification of reactivity could be the direct result of amino acid oxidation or an indirect result caused by a perturbation of the protein structure at the active site. The products oxidized at pH 8 lose their ability to react with H2O2. The products oxidized at pH4 react with H2O2 but their reactivity toward Fe(CN)4-6 is substantially reduced.


A kinetic study of the endogenous reduction of the oxidized sites in the primary cytochrome c peroxidase-hydrogen peroxide compound. The primatry compound formed in the reaction between H2O2 and cytochrome c peroxidase is oxidized two equivalents above the native enzyme. The two oxidized sites are thought to be an Fe(IV) and an amino acid radical. In the absence of oxidizable substrate, the Fe(IV) and radical sites decay by apparent first-order processes but at different rates. It is likely that the decay involves both intra- and intermolecular electron-transfer reactions. The reduction of the Fe(IV) site depends upon the pH with a minimum reduction rate of 2.9-10(-5)s(-1) at pH 6. At pH 4 and 6, the reduction of the Fe(IV) site is facilitated by prior oxidation of amino acid residues in the protein.


Evidence for the existence of a low spin complex in acidic methemoglobin: its structure and formation. By means of electron spin resonance and magneto-optical rotation, specific low spin complexes in acidic methemoglobin are obtained. The formation of these complexes is explained by a specific stereochemical arrangement of the distal histidine in the absence of allosteric effectors inducing the formation of a low spin ligand at room temperature. At low temperature, however, the distal histidine is directly bound to the heme iron. As the formation of the low spin complexes depends on allosteric effectors it is suggested that via the distal histidine the affinity of heme iron ligands is modified.


Recombination of carbon monoxide with hemoglobin after flash photolysis of the carboxyderivative in mixed solvents at subzero temperature. The kinetics of recombination of carbon monoxide to hemoglobin produced by total flash photolysis of its carboxyderivative are studied at low temperatures (down to --55 degrees C) in mixed hydroalcoholic solvents. The rates are found to be different in two solvents used, namely ethylene glycol/buffer and methanol/buffer; for the former, the rates at subzero temperatures are simply explained by cooling and are consistent with the activation energy as measured in aqueous solution, while those in methanol/buffer show evidence of a specific solvent effect. Values are reported for the rate constants and activation energies in the two solvents.


Analytical isotachophoresis in capillary tubes. Analysis of hemoglobin, hemiglobin cyanide and isoelectric fractions of hemiglobin cyanide. The zone stabilization in capillary isotachophoresis in the water phase has been improved by methylcellulose so that proteins can be analysed. Hemoglobin and hemiglobin cyanide samples were studied as model systems. Ampholine carrier ampholytes were used as spacers, enhancing the detection of the different components. The optimal amounts of Ampholine, however, were found to be much smaller than in most of the previously published reports. Linear relationships were found between the zone lengths and sample amounts, including spacers. The separations were reproducible and reached the isotachophoretic steady state. The hemiglobin cyanide was fractionated by isoelectric focusing. The four main fractions were then analyzed by capillary isotachophoresis and shown to be heterogeneous in mobility with a pH of 7.5 in the leading electrolyte. The component zones of the total hemiglobin cyanide sample were all identified in relation to the isotachophoretic components of the isoelectric fractions. The total analysis time was in average 30-40 min. The sample amounts were about 40 mug protein in each experiment with very small Ampholine volumes, 25-100 nl 40% (w/v).


The luminescence properties of concanavalin A. 1. The luminescence properties of native concanavalin A, both at room temperature and at 77 degrees K, are similar to those of other proteins containing tyrosine and tryptophan. 2. Binding of methyl alpha-D-glucopyranoside to concanavalin A causes a slight reduction of its fluorescence at room temperature. 3. Removal of Mn2+ and Ca2+ ions from concanavalin A causes a small increase in its fluoresence. The fluorescence: phosphorescence ratio and phosphorescence lifetime of apo-concanavalin A are similar to those of tryptophan. 4. Denaturation of concanavalin A by urea and by guanidine hydrochloride apparently takes place in two stages. Apo-concanavalin A is more easily denatured than the native molecule, but concavalin A combined with methyl alpha-D-glucopyranoside is more resistant to denaturation. 5. The luminescence properties of concanavalin A are pH-dependent. 6. The results have been interpreted in terms of the known structure and properties of concanavalin A.


The low-temperature luminescence properties of bovine alpha-lactalbumin. The luminescence of bovine alpha-lactalbumin at 77 K has been studied and compared with that of lysozyme. Alpha-Lactalbumin has several unusual properties, including a fluorescence spectrum showing vibrational fine structure, an abnormal phosphorescence spectrum, a high fluorescence: phosphorescence ratio and an abnormal phosphorescence decay. These properties are largely due to the proximity of tryptophan residues to disulphide bonds. Reduction of all these bonds causes considerable changes in alpha-lactalbumin luminescence, as does denaturation in acid solution. Reduction of a single labile disulphide bond has little effect, and the properties of alpha-lactalbumin III, a variant lacking one disulphide bond and one trypotophan residue, are similar to those of the normal protein. Several differences between alpha-lactalbumin and lysozyme are reported. The results support the suggestion that the two tryptophan residues found in the active site cleft of alpha-lactalbumin may be largely responsible for its luminescence.


Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions. The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.


Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process. In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.


In vivo and in vitro aging of collagen examined using an isometric melting technique. 1. In vivo and in vitro aging of tendon from rat tail, kangaroo tail and human wrist tendon was examined by the technique of isometric melting, in physiological saline. 2. For all these collagens, two mechanisms of structure stabilisation can be distinguished in the melting curves. One of these involves co-valent cross-linking as judged by its increasing stability to heat and acid pH, while the second appears to involve only secondary interactions. 3. The time rate of the first process is slow in vivo; rat tendon up to 2 years does not show it, but it is present in 6-year-old human tendon. However, its in vitro rate is markedly dependent upon the free oxygen content of the physiological saline. At an oxygen concentration of 300 nmol/ml, the in vitro aging rate is about 30 times the in vivo rate for rat tail tendon, and about 20 times for both kangaroo tail tendon and human wrist tendon. At a concentration of 60 nmol/ml (which is about the same as normal arteriovenous blood difference) in vitro aging proceeds close to the in vivo rate.


State and reactivity of tryptophyl residues in two bacterial proteases from Sorangium sp. The state and reactivity of tryptophyl residues in two proteolytic enzymes from Sorangium sp. were investigated by means of the following methods: spectrophotometric oxidation of tryptophans with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, and H2O2 in dioxane, optical rotatory dispersion, ultraviolet difference spectrophotometry, solvent perturbation and viscosity measurements. Out of two tryptophyl residues/molecule of alpha-lytic protease, one appears to be completely buried, while the other seems to be exposed. None of these two residues seem to be responsible for the activity of the enzyme. The beta-lytic protease undergoes an irreversible conformational transition between pH 5.0 and 3.5. Out of total four tryptophyl residues/molecule, only one is fully exposed at neutral pH. The other three are gradually exposed in the pH transition region. The degree of exposure and the dimensions of "cavities" shielding tryptophyl residues were estimated. The tryptophyl residues of of beta-lytic protease do not seem to participate in substrate binding or the active site; they are rather one of the determinants of the conformational state of the enzyme.


Salt effects on the denaturation of DNA. IV. A calorimetric study of the helix-coil conversion of the alternating copolymer poly[d(A-T)]. The enthalpy deltaH, entropy deltaS, and the temperature Tm of the conformational transition of poly[d (A-T)] from the ordered to the randomly oriented state have been determined at pH 6.8 with the help of an adiabatic differential scanning calorimeter in Na2SO4 solutions of increasing ionic strength. Spectrophotometric denaturation experiments supplemented the calorimetric measurements. All thermodynamic parameters were found to vary strongly with salt concentration: both deltaH and Tm increase linearly with the logarithm of the mean molal activity alpha plus or minus of Na2SO4. However, whereas the dependence of Tm on salt activity remains linear over the entire salt concentration range employed deltaH decreases abruptly in the most concentrated Na2SO4 solutions. The entropy of melting changes with salt concentration in a pattern similar to that displayed by deltaH. The data on deltaH as well as data derived from the maximum slopes of the calorimetric heat denaturation curves were used to calculate the cooperative length Lh, the stacking free energy epsilon, and the cooperativity parameter sigma of poly[d(A-T)] as a function of ionic strength. Lh decreases with increasing salt concentration whereas sigma increases. Epsilon assumes more positive values with increasing salt molality. These changes then are in agreement with the generally held belief that an increase in salt concentration leads to an increase in the "loop" content of the copolymer.


Specific conversion of s4 U to U in Escherichia coli tRNA by iodate oxidation. The minor nucleoside 4-thiouridine in Escherichia coli tRNA is transformed selectively to uridine by iodate oxidation at acidic pH. The four major nucleotides were found to be inert under these conditions. The iodate oxidation appears to be more specific than the previous conversion methods reported, and has the advantage that it does not affect the chargeability of most tRNA.


Puridication and properties of an intracellular ribonuclease from Candida lipolytica. 1. A ribonuclease (RNAase CL) (EC 3.1.4.23, ribonucleate 3'-oligonucleotide hydrolase) was extracted by EDTA/acetate buffer, pH 5.6 from acetonedried cells of Candida lipolytica and purified 1350-fold by acetone and (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography. 2. RNAase CL is an acidic protein having an isoelectric point of 4.2, and an approximate molecular weight of 32 000. 3. Optimal pH and temperature for the enzyme were 6.0 and 60 degrees C, respectively. It is stable at neutral pH up to 50 degrees C. At 64 degrees C for 30 min, 95, 49 and 64% inactivation of the enzyme occurred at pH values 4.2, 6.6 and 10.0, respectively. 4. RNAase CL inhibited by Zn2+ and Cu2+, sulfhydryl reactants and by high concentration of salts, but not by chelating agents. 5. RNAase CL degraded ribosomal RNA, transfer RNA, polyadenylic acid, polycytidylic acid and polyuridylic acid into acid-soluble nucleotides. Among the synthetic homopolymers, polycytidylic acid was most rapidly degraded. Polyguanylic acid and duplexes of synthetic homopolymers were less sensitive. DNA was not attacked. Specificity studies showed that RNAase CL preferentially cleaves pC-purine bonds. 6. Digestion of poly (C) by RNAase CL resulted in the liberation of cyclic 2',3'-CPM from the start of the reaction with no observable formation of intermediate oligonucleotides. This suggests that the enzyme depolymerizes by an exonucleolytic mechanism.


Histone-like protein in the prokaryote Thermoplasma acidophilum. The DNA of the prokaryote Thermoplasma acidophilum is associated with a histone-like protein that has the following properties: it has a high content (23%) of basic amino acids, is positively charged at neutral pH, is soluble in acid, and can stabilize DNA against thermal denaturation. In polyacrylamide gel electrophoresis, in the presence of either sodium dodecylsulfate or urea, it migrates at the same rate as histone IV (F2a1) of calf thymus. The amino acid composition, however, it unusually rich in the amides of acidic amino acids (16-20%), and it does not appear to be closely homologous to any of the classes of eukaryotic histones. Escherichia coli DNA, on the other hand, was associated with no detectable acid-soluble proteins, and the nucleoprotein thermally denatured at a lower temperature than pure DNA.


Effect of hydrogen ion concentration on energy metabolism in pig platelets. Respiration and glycolysis of pig platelets suspended in a dialyzed plasma were studied at various hydrogen ion concentrations. Respiration of platelets was high at acidic pH and decreased at physiological pH. This pH profile may not be attributed to properties of mitochondria, since the respiratory rate of mitochondria prepared from platelets was maximal at physiological pH. A low respiratory rate at physiological pH seemed to be attributable to depression of respiration by glycolysis, since the addition of glucose further depressed the rate. The Crabtree effect was more prominent at alkaline ph. glycolysis increased with an increase in the pH of the plasma, contrary to oxygen comsumption. The Pasteur effect was less prominent at alkaline pH. The effect of pH on lactate production by the cytosol fraction of platelets was similar to that of whole platelets. The glycolytic intermediate pattern showed that phosphofructolinase was the committed step. Both ATP concentration and ATP formation calculated from respiratory and glycolytic rates were constant at various pH values. These observations may indicate that the pH primarily affects platelet glycolysis at the phosphofructokinase step and the respiration is secondarily controlled by glycolysis.


Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin. 1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin. 2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca-2+ for aequorin and at least 3Ca-2+ for obelin. 3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units. 4. Other monovalent cations, such as Na+ are able to compete with Ca-2+ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions. 5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca-2+ solutions or of calcium/EGTA buffers.


Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts. Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.


Multichannel microspectrofluorometry for topographic and spectral analysis of NAD(P)H fluorescence in single living cells. Starting from a previously described prototype microspectrofluorometer a more versatile apparatus has been developed with rapid optional operation on a topographic mode for the simultaneous multisite evaluation of NAD(P) reduction-reoxidation transients or on a spectral mode for the analysis of natural and exogenous fluorochromes, in single living cells. On the topographic mode, adetailed kinetic analysis of NAD or NAD P-linked dehydrogenases can be made from 50-100 cell points imultaneously via automatic recording of topographic scans upt to 16 times a second, in correlation with microelectrophoretic intracellular inuection of metabolites (e.g. nearly immediate response to glucose 6-phosphate, 20-25 s delay for 6-phosphogluconate). Rapid shifts from topographic to spectral operation make possible the detection of a change in fluorescence intensity at a specific intracellular site and the immediate verification of its nature (NAD(P)H or exogenous fluorochrome) by spectral observations.


Activation of bovine liver glycerol kinase by ethanol. The crystallization of bovine glycerol kinase (ATP:glycerol 3-phosphotransferase EC 2.7.1.30) is reported along with a study on the unusual activation of this enzyme by ethanol. The enzyme was extracted from calf lever andusual activation of this enzyme by ethanol. The enzyme was extracted from calf liver and purified 5900-fold giving crystals with a 5% yield. The kinetics of the enzyme with regard to glycerol and ATP were studied by varying the concentration of one substrate while keeping the other at saturating levels, and the effect of ethanol was observed by adding it at levels of 5% (v/v). Ethanol increased the V in both cases almost 2-fold. The apparent Km of ATP was 3.5 - 10(-6) and was increased to 7.6 - 10(-6) in the presence of ethanol. The apparent Km for glycerol was 3 - 10(-5) and was increased to 12 - 10(-5) when ethanol was added. A number of other alcohols had a similar activating effect except for 1,2-diols which only inhibited the enzyme. These findings are consistent with the explanation that alcohols compete with glycerol (hence also with the glycerophosphate product) for a hydroxy binding site on the enzyme. This leads to more rapid dissociation of the glycerophosphate (i.e. an increase in the steady-state constant, "k+2" resulting in an increased V).


Protamine kinase from yeast.

Protamine kinase from yeast. A protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) which preferentially phosphorylates protamine is purified about 250-fold from the soluble fraction of baker's yeast (Saccharomyces cerevisiae). This enzyme is not sensitive to activation by cyclic nucleotides. Histone is about 5% as active as protamine in the reaction rate. Neither casein, phosvitin nor glycogen phosphorylase is active as substrate. The enzyme is distinguishable from casein kinase of the classical type (Rabinowitz, M. and Lipmann, F. (1960) J. Biol. Chem. 235, 1043-1050) and from adenoshine 3', 5'-monophosphate-dependent protein kinase described earlier (Takai, Y., Yamamura, H. and Nishizuka, Y. (1974) J. Biol. Chem. 249,530-535).


3':5'-cyclic-nucleotide phosphodiesterases of mammalian sera. Cyclic AMP and cyclic GMP phosphodiesterases (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) were found in the sera of human, dog, rabbit and rat. The formed elements of blood were not present in sera and thus not the source of the phosphodiesterase. More rapid hydrolysis of cyclic GMP than cyclic AMP is observed in the sera of these four species when 0.4 muM of cyclic AMP or cyclic GMP is used as the substrate. Protein activator of the phosphodiesterase is not detectable in the sera of these four species. Serum cyclic AMP and cyclic GMP phosphodiesterase activities are not stimulated by protein activator prepared from bovine brain. The serum phosphodiesterases of these four species are purified through Sepharose 6B column chromatography. Cyclic AMP phosphodiesterase are found in a broad area corresponding to molecular weights ranging from approximately 150 000 to 340 000 with 2 to 3 peaks in all animals tested. Cyclic GMP hosphodiesterase is found in a single area corresponding to molecular weights of 230 000 (rabbit and rat) and 270 000 (human and dog). Serum cyclic AMP and cyclic GMP hosphodiesterase activities of these four species have pH optimum of 7.5-8.5. Optimal concentration of Mg-2+ is about 5 mM for cyclic GMP phosphodiesterase activities of these four species as well as for cyclic AMP phosphodiesterase activities except rabbit. Rabbit serum cyclic AMP phosphodiesterase requires higher concentration of Mg-2+ (50 mM) for its optimal activity. The double reciprocal plots are non-linear for cyclic AMP phosphodiesterases of all animals and cyclic GMP phosphodiesterases of human, dog and rat. Rabbit cyclic GMP phosphodiesterase exhibits  a linear reciprocal plot. Cyclic GMP is inhibitor of serum cyclic AM phosphodiesterase. Rabbit enzyme was most effectively inhibited by cyclic GMP.


Characterisation of cyclic adenosine 3':5'-monophosphate phospodiesterase from Walker carcinoma sensitive and resistant to bifunctional alkylating agents. Walker carcinoma cell lines sensitive or resistant to bifunctional alkylating agents have been found to contain multiple forms of cyclic AMP phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). These activities have been resolved using Sepharose 6B gel filtration and their apparent molecular weights have been estimated. The enzyme appears to occur in four active forms of apparent mol. wts of greater than 1 000 000, 430 000, 350 000 and 225 000, when assayed at low substrate concentrations. Evidence has been obtained which suggests that all four forms of the enzyme are composed of subunits of mol. wt of approximately 15 000 and are interconvertible. While the ionic strength of the buffer affected the predominance of the different forms, the presence of cyclic AMP at 10(-6) M had no effect on aggregation or dissociation of the enzyme. An activity shift from high molecular weight forms of the enzyme to low molecular weight forms has been found in the resistant tumour at low substrate concentration. No change in elution profile between sensitive and resistant tumours was observed for the low affinity form of the enzyme. The pH optima of the enzymes with both high and low affinity for the substrate was found to be pH 8.0 in the sensitive line. In the resistant tumour the pH optima of the high affinity form is shifted to pH 8.4 while the low affinity form remains at pH 8.0. The high affinity forms of the phosphodiesterase in the sensitive and resistant tumour also differed in their inhibition by theophylline. In both cases inhibition was of the competitive type with Ki values for the sensitive and resistant lines being 2.35 and 0.32 mM, respectively. There was no significant difference in the inhibition of the low affinity form between the sensitive and resistant tumour.


Intracellular distribution of beta-galactosidases in mucosal cells from hog small intestine. 1. Intracellular distribution of three types of beta-galactosidases (beta-D-galactoside galactohydrolase EC 3.2.1.12) i.e. hetero beta-galactosidase, lactase and acid beta-galactosidase, was studied by examining the properties of subcellular fractions isolated by a systematic fractionation of mucosal cells of the small intestine of the hog. Localization of hetero beta-galactosidase in cytosol could be shown. 2. Localization of lactase in the brush borders was shown by analyzing the purified brush borders prepared separately. 3. To domonstrate the lysosomal localization of acid belta-galactosidase, lysosomes were purified separately and their extract was chromatographed on a hydroxylapatite column. The activities of various enzymes in the purified lysosomes as well as in the intermediary fractions obtained during lysosome purification and the pattern of the hydroxylapatite chromatography led us to conclude that acid beta-galactosidase is a lysosomal enzyme.


Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism. Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.


Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase. K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.


Oxidation of dicyano-bis(1,10 phenanthroline) iron(II) by compounds I and II of cytochrome c peroxidase. The apparent bimolecular rate constant for the oxidation of dicyano-bis(1,10 phenanthroline) iron(II) by compound II of cytochrome c peroxidase (ferrocytochrome c; hydrogen-peroxide oxidoreductase EC 1.11.1.5) has been measured over the pH range 2.5-11.0 at 0.1 M ionic strength, 25 degrees C, by the stopped-flow technique. An ionizable group in the enzyme, with a pKa of 4.5, strongly influences the electron transfer rate between the ferrous complex and the oxidized site in the enzyme. The electron transfer is fastest when the group is protonated, with a rate constant of 2.9 - 10-5 M--1 - s-1. The rate constantdecreases over three orders of magnitude when the proton dissociates. The apparent bimolecular rate constant for the oxidation of the ferrous complex by compound I of cytochrome c peroxidase was determined between pH 3.5 and 6. Under all conditions where this rate constant could be measured it was about three times larger than that for the oxidation by compound II.


A calcium ion-dependent atp pyrophosphohydrolase in Physarum polycephalum. An activity of ATP Pyrophosphohydrolase (EC 3.6.1.8) was found in the soluble fraction of the plasmodium of Physarum polycephalum. The products of the enzyme reaction were inorganic pyrophosphate and 5'-AMP in equimolar quantities. The enzyme had a pronounced requirement for Ca2+ with high specificity. Mg-2+ was not an essential cofactor but stimulated the enzyme activity about 2.5-fold of the control. The enzyme hydrolyzed ITP, GTP and beta,gamma-methylene ATP at a limited rate. Among inhibitors tested, 3 mM caffeine reduced the activity to about 75% of the control. The enzyme had a broad pH optimum around pH=7.0 and the Km for ATP was 2.0 mM. An Arrhenius plot showed a break at about 18 degrees C and the calculated activation energies were 6.7 and 11.4 kcal/mol above and below the transition temperature, respectively. Disc electrophoresis in dodecyl sulfate and gel filtration on Sephadex -g-200 gave apparent molecular weights of 56 000 and 240 000, respectively, suggesting that the native enzyme was built up from 4 polypeptide chains. The possible role of the enzyme in vivo was discussed.


Further studies on tryptophan hydroxylase from neoplastic murine mast cells. Tryptophan hydroxylase (tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4) purified from the neoplastic murine mast cells by hydroxylapatite chromatography following ammonium sulfate fractionation showed maximum activity at pH 6.0 in the presence of 2-mercaptoethanol, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine and Fe2+, and pH 7.6 to 8.0 in the absence of addED Fe2+. The Km values were 38.5 muM and 22.2 muM for tryptophan, 298 muM and 204 muM for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine, and 6.45% for oxygen in either presence or absence of added Fe-2+, respectively. From kinetic data the reaction mechanism of tryptophan hydroxylation appears to be of the sequential, rather than the ping-pong, type. Tryptophan hydroxylase from mast cells was considerably inhibited by o-phenanthroline like phenylalanine hydroxylase as well as tyrosine hydroxylase from other sources, and its Ki was between 1.2 muM and 4.53 muM. It was found that the inhibition by o-phenanthroline was competitive with respect to both tryptophan and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not molecular oxygen under the assay conditions employed.


Purification and characterization of two activities of the intracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299. Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5) activity of Leuconostoc mesenteroides NRRL B-1299 cells has been purified by adsorption on hydroxyapatite, followed by chromatographies on DEAE-cellulose and DEAE-Sephadex. The enzyme activity was readily separated into two principal forms of the enzyme, I and II, by DEAE-cellulose chromatography. Both enzymes I and II were purified to an electrophoretically homogeneous state in which the relative enzyme activities reached 32-and 14-fold of the original specimen, respectively. Molecular weights were 69 000 for the enzyme I and 79 000 for the enzyme II as determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide, and no subunit structure was observed. Enzyme I had an optimum pH at 6.3-6.5 and exhibited a maximal activity at 45 degrees C, while the optimum pH and temperature of enzyme II were pH 5.5-5.9 and 35-40 degrees C. The Km values of enzymes I and II were 10.7 and 250 mM, respectively. The effects of several metal ions, chemical reagents, and addition of various dextrans were also examined. Beside linear alpha-1,6-linkages the polymer synthesized by the enzyme II contained lesser amount of alpha-1,2-and alpha-1,3-linkages, which seems to be a primary characteristic of the B-1299 dextran.


Regulation of hepatic tyrosine aminotransferase in the frog Rana temporaria. The regulation of hepatic tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) in the rat has been extensively studied but little is known about the enzyme from other sources. We have studied the regulation of this enzyme in the frog Rana temporaria and in this paper we report that: 1. Cortisone acetate, adrenocorticotropic hormone and alpha-methyl-p-tyrosine, an agent known to induce hepatic tyrosine aminotransferase in the rat via activation of the pituitary-adrenal axis, have no effect on the activity of the enzyme in the frog. 2. Dibutyryl-3',5'-cyclic AMP induces the enzyme to about 2-fold. 3. Injection of tyrosine methyl ester and a protein-rich diet result in an increase in the enzyme activity. This increase is of the same order of magnitude as that caused by dibutyryl cyclic AMP. 4. Glucose significantly reduces tyrosine aminotransferase activity in frog liver. These results suggest that cyclic AMP induces the enzyme via a mechanism independent of glucocorticoids. The frog offers a model for studies on the regulation of hepatic tyrosine aminotransferase in vivo without interference from secondary effects mediated by the adrenals.


Enzymic reactions of fatty acid hydroperoxides in extracts of potato tuber. I. Comparison 9D- and 13L-hydroperoxy-octadecadienoic acids as substrates for the formation of a divinyl ether derivative. 1. 9-D-Hydroperoxy-octadeca-trans-10, cis-12-dienoic acid (formed from linoleic acid by potato lipoxygenase) is enzymically converted to a divinyl ether derivative (colneleic acid) by extracts of potato tuber. 2. the isomeric 13-L-hydroperoxide (formed from linoleic acid by soyabean lipoxygenase) is not a substrate for this enzyme system. 3. the enzyme reaction was not dependent on oxygen and was unaffected by metal chelators and thiol reagents. 4. studies with 18O-labelled substrate indicate that the ether oxygen atom in colneleic acid is not derived from the oxygens of the hydroperoxide group.


Preparation of 32P-labeled inositides and their degradation by soluble kidney enzymes. A method is described for the preparation of radioactive inositol lipids for studies of their enzymic degradation. Kidney cytosol fractions have been used to produce diesteratic cleavage. High voltage electrophoresis at pH 4.3 is used to separate D-myoinositol 1 : 2-cyclic phosphate and D-myoinositol 1-phosphate from hydrolysis of phosphatidylinositol. Radioactivity co-migrating with myoinositol diphosphate and triphosphate is separated by electrophoresis at pH 1.5 following enzymatic hydrolysis of phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Relative activities for hydrolysis of the various inositides suggest the presence of more than one phosphodiesterase.


Ceramidase and ceramide synthesis in human kidney and cerebellum. Description of a new alkaline ceramidase. It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.


Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver. The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.


Partial purification and properties of phospholipase A2 from the starfish, Asterina pectinifera. 1. Phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) activity was shown to occur in the supernatant fraction of a freshly prepared homogenate from the pyloric caecum of starfish (Asterina pectinifera). 2. The phospholipase A2 has been isolated and purified 130-fold by ultracentrifugation, ammonium sulfate precipitation and column chromatographic procedures. 3. The purified enzyme was stable to heat at low pH values and the optimal pH was observed at approximately 9.0. 4. The enzyme activity was activated by Ca2+ and sodium deoxycholate, and was inhibited by EDTA.


Studies on the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipid. The importance of metal chelation in the mechanism of microsomal lipid peroxidation has been studied using both phosphate- and sulfhydryl-containing compounds. The optimal concentration for maximum stimulation by each of these compounds has been determined, and the decrease in stimulation observe at concentrations above the maxima has been related to the ability of these compounds to form stable chelation complexes with non-heme iron. Of the compounds tested, only ADP and ATP facilitated the cooperative binding of NADPH to the membrane and thus suggested the possibility of three binding sites for NADPH. Neither of the other two phosphate-chelating agents (Pi or PPi) and neither of the two thiols (cysteine or dithiothreitol)facilitated cooperative binding of NADPH. These data suggested that the adenine ring of ADP or ATP is directly involved in the cooperativity of NADPH binding. They also emphasized that the binding of the chelation complex to the protein is an important parameter in the mechanism of the NADPH-catalyzed peroxidation of endogenous microsomal lipids. Furthermore, stimulation of the rat of lipid peroxidation by sulhydryl-containing compounds, by freezing thawing the microsomal protein, and by treatment of the protein with detergent may be due to a decrease in this cooperative binding effect. Since cysteine and deoxycholate as well as freezing and thawing alter membrane structure, the stimulation of lipid peroxidation seems to involve some alteration to the structure of the microsomal membrane prior to the onset of enzymatic lipid peroxidation.


The substrate for cholesterol 7alpha-hydroxylase. The rates of incorporation of (14C) cholesterol into cholesteryl esters and 5-cholestene-3beta,7alpha-diol (7alpha-hydroxycholesterol) by rat liver microsomes, measured under conditions in which esterification and 7alpha-hydroxylation are varied independently, indicated that cholesterol is the substrate for cholesterol 7alpha-hydroxylase. The specific activities of cholesteryl esters and 7alpha-hydroxycholeste  ol in incubations of microsomes labelled with (14C)cholesterol in vitro or in vivo suggest that 7alpha-hydroxycholesterol and esterified cholesterol are not derived from the same pool of free cholesterol.


Some characteristics of the prostaglandin synthesizing system in rabbit kidney microsomes. The prostaglandin synthesizing system in rabbit kidney microsomes was characterised using a radiometric assay. Three prostaglandins (F2alpha, E2 and D2) were formed form (1-14C)arachidonic acid, a small amount of prostaglandin A2 was also detected but this was formed non-enzymatically. Biosynthesis was stimulated by reduced-glutathione and 1-adrenaline and was inhibited by aspirin-like drugs. The enzyme system was sensitive to small changes in pH. There were substantial differences in drug sensitivity and optimal reaction conditions between this prostaglandin synthesizing system and the one from bovine seminal vesicles.


Glutathione biosynthesis in Escherichia coli K 12. Properties of the enzymes and regulation. The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts. The pH optima and the apparent Km values for the substrates have been determined for both synthesizing enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase. gamma-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase. In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 mumol per ml cell pellet in the stationary growth phase. GSSG was not detectable. The levels of the enzymes remained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids. The pathway is controlled by feedback inhibition and not by repression.


The characterisation and function of the polysaccharidases of human synovial fluid in rheumatoid and osteoarthritis. A potential enzymic mechanism for the degradation of glycosaminogly cans was characterised using enzymes found in rheumatoid synovial fluid from the knee joint. This mechanism involves a true hyluronidase together with the concerted action of beta-glucuronidase and beta-N-acetylhexosaminidase. The contribution of the exopolysaccharidases to hyaluronate degradation was demonstrated by the use of specific inhibitors, while the distinct identity of a true hyaluronidase was shown by ammonium sulphate and agarose gel column fractionations. Only the hyluronidase fraction was capable of degrading high molecular weight hyaluronate. The exopolysaccharidase activities were shown to be markedly elevated in rheumatoid as compared to osteoarthritic synovial fluid and also normal serum. On the other hand, hyluronidase was similarly active in rheumatoid and osteoarthritic synovial fluids; both these levels were lower than that of normal human serum. Hyaluronidase in synovial fluid may thus be derived by diffusion from serum, since it is of relatively low molecular weight (60 000). The pH requirements of this enzyme system and the strong inhibition of hyaluronidase by synovial fluid make it unlikely that the mechanism operates extracellularly. It is proposed that as a lysosomal mechanism, however, it is an important contributing factor in the chronic erosion process characteristic of rheumatoid arthritis.


Human umbilical cord hyaluronate. Neutral sugar content and carbohydrate-protein linkage studies. Paper chromatography of neutral sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neutral sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced eith NaBH4 -PdCl2, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular wieght to about one-half and an appreciable decrease in the specific rota tion of hyaluronate, suggesting a separation of the two antiparallel chains o the double helical hyaluronate. The umbilical cord hyluronate containe contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.


Characterization of a colchicine receptor protein in rat epididymal adipose tissue. Colchincine was found to be taken up by adipose tissue and therein to bind to a soluble macromolecule not sedimented by centrifugation for 2 h at 100 000 x g. A similar binding occurred when soluble extracts of adipose tissue were incubated with colchicine. The binding reaction reaction is temperature dependent and shows a pH optimum between 6.8 and 7.0. Double reciprocal plots of colchicine concentration versus amounts of colchicine bound to protein in the steady state disclosed an apparent Km of 0.250 to 1.5 muM. The colchicine binding activity of soluble tissue extracts decreased when the extracts were incubated at 37 degree C. Addition of guanosine triphosphate and Mg-2+ retarded the loss of colchicine binding activity. The molecular weight of the colchicine complex was estimated to be 115 000 and its sedimentation coefficient 5.8 S. All of these characteristics are remarkably similar to those of the protein tubulin which has been isolated from other tissues. Since it is now well known that tubulin is a protein subunit of cytoplasmic microtubules, it is suggested that the previously reported metabolic effects of colchicine on adipose tissue result from the dissolution of microtubules by colchicine.


Interaction of nerve growth factor with tubulin. Studies on binding and induced polymerization. The interaction of the nerve growth factor with the neurotubule protein has been studied with the aim of elucidating the nature of the large complexes that they form when incubated together and the factors and control this event. The results show that the binding of nerve growth factor to tubulin is followed by the formation of large structures that, in certain experimental conditions, accelerate the rate of tubulin polymerization to form microtubules or catalyze their assembly in conditions where this process does not occur spontaneously. The formation of large nerve growth factor-tubulin complexes starts to occur only at a molar ratio of 1.0-1.5 NaCL or GTP strongly inhibit this proceed without a detectable effect on NGF binding. Two hypotheses are postulated explain these findings. Firstly, that tubulin has two sites with different affinity for nerve growth factor and the polymerization occurs only when the second NGF molecule has interacted with the microtubule protein. Alternatively, free tubulin in solution is the polymerization by hindering site of tubulin-factor complexes present in solution at a 1.1 molar ratio. In both cases, GTP, Na-+ or H-+ will affect the formation of large unsoluble, tubulin-NGF complexes, by changing their conformation or by decreasing electrostatic interactions.


Investigation of some physico-chemical properties of muscular parvalbumins by means of the luminescence of their phenylalanyl residues. The influence of pH, temperature and Ca2+-release on the phenylalanyl and tyrosyl fluorescene of muscular parvalbumins from white muscles of hake and carp has been investigated. Within the pH range from 7 to 8, Ca2+-saturated parvalbumins show a conformational change registered by fluorescence, that is associated with the release of some of the bound Ca2+. Removal of Ca2+ by means of EGTA (ethyleneglycolbis-(aminoethylether)tetra-acetic acid) considerably narrows the region of protein nativity, increases the accessibility of their chromophores to quencher ions (Cs+ and CNS-) and decreases their stability against heat denaturation. The usefulness of measurements of the phenylalanine fluorescence and of the tyrosine-phenylalanine energy transfer in the investigation of these and other proteins is discussed.


NMR studies of hemoproteins. VI. Acid-base transitions of ferric myoglobin and its imidazole complex. 220 MHz proton NMR was applied to the acid-base transition of ferric myoglobin and its imidazole complex. In horse and sperm whale ferric myoglobins: (1) pH-dependent shift of heme-ring methyl signals above p2H 10 was analyzed on the basis of rapid exchange between alkaline and acidic forms by the use of pK value 9.1 of acid-base transition in 1H20 solution; (2) limiting shifts of three methyl signals were reasonably determined for purely alkaline form. For the imidazole complex: (3) a drastic high field shift of each signal was observed above p2H 9.0, whereas N0methyl imidazole complex did not exhibit such a shift, which suggests the 2H+ dissociation from liganded imidazole greater than N2H. It is concluded thns.


Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus). A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.


Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution. A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4-5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes posses mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.


Interaction of troponin components with F-actin and F-actin-tropomyosin complex. 1. Both TN-T and TN-I components of troponin interact with F-actin, causing its precipitation at 0.1 M KC1 and neutral pH in a form of highly ordered paracrystals, although the ability of TN-I component to precipitate of F-actin is much weaker. 2. F-actin paracrystals obtained in the presence of both TN-T and TN-I components consist of parallel arrays of F-actin filaments, although the fine structure is in each case different. 3. In the presence of tropomyosin in the proportion equal to that in muscle, less TN-T or TN-I component is needed to obtain full precipitation of F-actin. 4. Paracrystals of F-actin-tropomyosin-TN-T component and F-actin-tropomyosin-TN-I component show regular transverse striation spaced at about 380 A intervals. 5. The TN-C component of troponin solubilizes all precipitates of F-actin with TN-T or TN-I components, regardless of the presence of tropomyosin. 6. The results show that both TN-T or TN-I components can bind independently to F-actin-tropomyosin complex with the same periodicity, similar to that of the whole troponin in the living muscle.


Interpretation of the electron spin resonance spectra of nitroxide-maleimide-labelled proteins and the use of this technique in the study of albumin and biomembranes. The composite ESR spectra of nitroxide-maleimide spin-labelled albumin and mitochondrial membranes were investigated. It was found that the mobile spectrum (so called "weakly immobilized") is due to the probe free in solution; it could be removed by dialysis, by lowering the ratio of spin label to albumin or by using acid pH values. Similar degrees of mobility were noted with spectra of spin label in solutions with viscosities similar to that of albumin. The immobile spectrum (so called "strongly immobilized") is due to the binding of N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide to the proteins. Studies with modified albumins have shown that in this binding the free SH and amino groups are involved, but the three-dimensional structure offering conditions for hydrophobic binding is also required. The spectral parameters useful for evaluating conformational changes of proteins and an application to mitochondrial membranes are described.


A polypeptide conjugate of bilirubin from human bile. An alkali-stable bilirubin conjugate has been obtained from human T-tube bile as its phenylazo derivative. The conjugate consists of a polypeptide probably of molecular weight 7000 to which the azo pigment of bilirubin is linked covalently through its carboxyl group. It thus constitutes the first biliprotein found in mammals. It is not known whether both carboxyl groups of native bilirubin participate in the binding of the conjugating protein, nor has it been possible to determine the number of pigment moieties occurring on a single polypeptide chain. The isolation makes use of the tendency of the conjugate to form large aggregates and involves the following steps: azo coupling of the native bile, (NH4)2S04 precipitation of macromolecules and aggregates, removal of low molecular weight contaminants by dialysis and gel filtration (first on Sepharose 6B IN 6 M guanidine, then on Sephadex LH-20 in 50% acqueous 2-chloroethanol) and a concluding purification by chromatography on p-aminobenzyl cellulose using a PH gradient. The final preparation appeared to be homogeneous on polyacrylamide electrophoresis.