Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate: production of an ammonia-containing intermediate in the "burst" phase. We have studied the effects of ammonium acetate on the transient "burst" phase of the oxidation of L-glutamate by glutamate dehydrogenase. Two measurable changes are observed in the "burst" phase as ammonium acetate concentration is increased: (i) an increase in the apparent first-order rate constant, kapp, and (ii) a decrease in the amplitude of the absorbance change measured at 320 nm. The increase in kapp shows a hyperbolic dependence on ammonium acetate concentration and is independent of glutamate concentration. The results demonstrate the existence of an intermediate immediately following hydrogen transfer. The intermediate contains enzyme, reduced coenzyme, ammonia, and alpha-ketoglutarate moieties and is in equilibrium with the known complex consisting of enzyme, reduced coenzyme, and alpha-ketoglutarate. At high concentrations of ammonium acetate, the equilibrium favors the ammonia containing complex.


Use of ethidium bromide fluorescence enhancement to detect duplex DNA and DNA bacteriophages during zone sedimentation in sucrose gradients: molecular weight of DNA as a function of sedimentation rate. Duplex DNA molecules and DNA bacteriophages have been sedimented through 5--25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DNAs from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0.38 when 0.025--0.70 microgram of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide--sucrose gradients and detected by fluorescence enhancement.


In vitro oxidation of intrinsic sulfhydryl groups yields polymers of the two predominant polypeptides in the nuclear envelope fraction. The two quantitatively predominant nuclear nonhistone polypeptides of the avian erythrocyte comprise a significant portion of the nuclear envelope protein. Each of these two polypeptides can be converted almost completely to homogeneous polymeric species via oxidation of intrinsic sulfhydryl groups by omicron-phenanthroline cupric ion complex. Major products are two dimeric species arising from each of the approximately 77 000-dalton pair. The larger of the two polypeptides also yields oligomers greater than dimers. These observations extend out earlier solubility and polypeptide cross-linking evidence which indicated that these polypeptides were involved in an oligomeric, proteinaceous nuclear structure. Ultramicroscopic studies from other laboratories indicate that such a structure may function as a nuclear skeleton. The architectural details of the structure now appear to be accessible at the molecular level.


Conformational states of a hydrophobic protein. The coat protein of fd bacteriophage. The coat protein of fd bacteriophage has a short polypeptide chain of only 50 amino acid residues, containing a highly hydrophobic segment of 19 amino acids that is entirely devoid of ionic or other strongly polar amino acids. In the viral particle the protein exists as a closely packed array of alpha helices. It can be transformed to a monomeric randomly coiled polypeptide in very concentrated (greater than or equal to 7.3 M) guanidinium chloride. In anionic detergents or phospholipids the protein is dimeric, with a mixed conformation ("50% alpha"), the hydrophobic segment having a beta structure, whereas the two ends are predominantly alpha helical. In guanidinium chloride at concentrations of 6 M or less, and under other conditions in the absence of an anionic detergent or phospholipid, the protein forms an intractable polymer, with a beta-type conformation. If the protein is succinylated an oligomeric form of this structure (speculatively thought to be a soluble variety of a "beta barrel") can be obtained as a metastable state. The 50% alpha conformation, the beta oligomer, and the random coil can be interconverted reversibly, but formation of the beta polymer appears to be irreversible.


High-pressure liquid chromatography in polynucleotide synthesis. Reverse phase high-pressure liquid chromatography (HPLC) using columns containing microparticulate materials with bonded octadecyl groups has been developed as a rapid and efficient method for the separation of nucleosides, nucleotides, and, in particular, of protected oligonucleotides which are standard intermediates in the stepwise synthesis of deoxyribopolynucleotides. Reported are extensive studies of the influence of the different purine and pyrimidine bases, of protecting groups, of the phosphate groups, and of the chain lengths of oligonucleotides on their retention on such columns. Further, the application of HPLC in the stepwise synthesis of an oligonucleotide, d(G-G-A-A-G-C-T-T-A-A-C), has been described. The methods, which are herein described, lend themselves to separations on a preparative scale and effect a marked reduction (up to 50%) in the time required for the synthesis of oligonucleotides.


Use of the lipophilic tert-butyldiphenylsilyl protecting group in synthesis and rapid separation of polynucleotides. Butyldiphenylchlorosilanes react with deoxy- and N-protected deoxymononucleotides to give, in each case, the 3'-O-butyldiphenylsilyl ether in high yield. A comparative study of n-, sec-, and tert-butyldiphenylchlorosilanes showed, as expected, a wide variation in the rate of formation and in the stability of the 3'-O-silyl ethers, the tert-butylphenylsilyl group being the one of choice in polynucleotide synthesis. The silyl group can be readily removed on treatment with fluoride ion in buffered pyridine at room temperature. This property markedly increases flexibility of the current methodology for polynucleotide synthesis. Secondly, because of its high lipophilicity, the silyl group allows rapid solvent extraction of synthetic intermediates, and thirdly, it greatly facilitates the isolation of the required product in synthetic reactions because of the selective and strong retention of the condensation product during reverse-phase high-pressure liquid chromatography. Several examples of synthetic procedures, including a stepwise synthesis of an undecanucleotide, are given which demonstrate overall simplification of polynucleotide synthesis.


Conformation of dinucleoside monophosphates modified with benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide as measured by circular dichroism. The conformational properties of GpU modified with the reactive derivative of benzo[a]pyrene, (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, has been investigated utilizing circular dichroism spectroscopy. Binding of this carcinogen to the N2 of G residues in GpU resulted in the formation of four compounds (I to IV) representing two pairs of diastereoisomers. The molar ellipticity values of the modified dimers were approximately twofold higher than those of the modified guanosine monomers. These values were decreased appreciably when the spectra of the dimers were obtained at 80 degrees C or in methanol rather than at 25 degrees C in water, suggesting that under the latter conditions there is a stacking interaction between the carcinogen and the neighboring uridine residue. Based on these results, a conformation is proposed for modified GpU. It includes insertion of the benzo[a]pyrene moiety, by rotation of the modified guanine residue about its glycoside bond, coplanar to the neighboring uridine and perpendicular to the phosphodiester backbone.


Mitochondrial phospholipase A2 activity and mitochondrial aging. The changes in mitochondrial phospholipid metabolism and energy-linked functions have been followed as coupled mitochondria are allowed to age in isotonic sucrose at 18 degrees C. Analysis of the aging process has provided an approach for studying the structure--function relationships within the mitochondrion without adding external agents to perturb the membrane structure. The initial event observed in this process of deterioration is a loss of respiratory control which is paralleled by diminishing levels of ATP. As ATP levels decline, so do the rates of reacylation of monoacyglycerophosphorylethanolamine and fatty acid oxidation. In most cases the previously inactive phospholipase A2 (EC 3.1.1.4, phosphatide-2-acyl-hydrolase) begins rapid hydrolysis of membrane phosphatidylethanolamine as ATP levels approach zero. The final energy-linked phenomenon observed to decline is the anilinonaphthalenesulfonic acid fluorescence response. Evidence is presented which suggests strongly that the activity of the mitochondrial phospholipase A2 on endogenous phospholipids is suppressed in tightly coupled mitochondria. This suppression is temporally linked to ATP levels in the mitochondria. Furthermore, this study demonstrates that mitochondria which are only slightly damaged have the potential to effect membrane repair through reacylation of monoacyl phospholipids.


Two human trypsinogens. Purification, molecular properties, and N-terminal sequences. The two human trypsinogens have been isolated from human pancreatic juice in a sufficient amount to study molecular and structural properties. The purification procedure included filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The two trypsinogens represent 19% of total proteins of pancreatic juice. Trypsinogen 1, the major form, is present in a quantity twice that of trypsinogen 2, which is the most anionic protein in human pancreatic juice. The two proteins have partial immunological identity, close molecular weights (23 438 and 25 006 for trypsinogens 1 and 2, respectively) and similar amino acid compositions. The N-terminal sequences are the same for the first 9 residues: Ala-Pro-Phe-Asp4-Lys-Ile. The two proteins differ in the activation peptides released during the transformation to trypsins. Trypsinogen 2 liberates one octapeptide Ala-Pro-Phe-Asp4-Lys while trypsinogen 1 liberates two peptides, the same octapeptide and the pentapeptide (Asp)4-Lys.


Bombesin, bombesin analogues, and related peptides: effects on thermoregulation. The synthesis and biological evaluation on thermoregulation of 39 peptides related to bombesin (structural analogues or other naturally occurring peptides) are described. The bioassay system reported measures the ability of peptides injected intracisternally to lower body temperature of cold (4 degrees C) exposed rats. The most potent analogues of bombesin were those in which positions one to five (not included) were altered, indicating that the decapeptide C terminal was sufficient for full potency. Gln at the seventh position and Gly at the 11th position could be replaced by D-Gln and D-Ala (but not D-Pro or D-Phe), respectively, without any change in potency. Methionine at the 14 position could be replaced with its D isomer with retention of 10% biological activity. Any other alteration of the C terminus (deletions or free acid with the exception of the N-methylamide) drastically reduced the biological potency of those peptides. Among other naturally occurring peptides, alytesin was found to have 100% of bombesin potency whereas litorin, neurotensin, xenopsin, substance P, physalaemin, and eledoisin were found to be in the order of 10(4) times less potent. The shortest peptide found to have full biological activity is the octapeptide des-Glp-Gln-Arg-Leu-Gly-Asn[D-Glp7, D-Ala11]-bombesin.


High resolution proton magnetic resonance spectroscopy of histones and histone--histone complexes in aqueous solution. Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.


Energy transfer and site of energy trapping in photosystem I. Studies of mutants of Chlamydomonas reinhardi deficient in the chlorophyll . protein complex CP1 show that energy transfer occurs between at least three Photosystem I units. Evidence for the connection of the Photosystem I units is also obtained by studying their optical cross-section in wild-type cells in which a fraction of the Photosystem I centers are inactivated. The site of energy trapping in the Photosystem I unit is shown to be the photochemical center itself and not the surrounding long-wavelength absorbing forms of chlorophyll.


Photoselection and aggregation in purple membrane of Halobacterium halobium. Preparations of the purple membrane of Halobacterium halobium suspended in dilute buffer and basal salt have been examined by circular dichroism spectroscopy and correlation analysis of scattered laser light. Dark adapted samples suspended in basal salt show photoselection when examined by circular dichroism. This was confirmed by irradiation with plane polarized light. Light-adapted samples or dark-adapted samples suspended in dilute buffer did not show this phenomenon. The reaction responsible for photoselection was shown to be the light induced cis-trans isomerization of bacteriorhodopsin. The stability of the induced anisotropy was due to aggregation in the basal salt suspensions which occurred despite little or no visual indication. This aggregation was confirmed by correlation analysis of scattered laser light.


Photosynthetic oxygen evolution from hydrogen peroxide. A study was made of the interactions of flash-illuminated chloroplasts with hydrogen peroxide. We conclude: 1. The oxygen precursor system can be reduced beyond the S0 state to an S-1 state, which can be oxidized to S0 by a single flash. 2. In the dark, a two-electron donation by H2O2 takes place which reduces S2 to S0 and S1 to S-1. 3. At the same time, two-electron oxidations by H2O2 re-form, S2 from S0 and S1 from S-1. 4. The catalase-like activity due to this cyclic oxidation and reduction of the S enzyme is higher with the S2 in equilibrium S0 couple than with the S1 in equilibrium S-1 couple. Another process, however, is responsible for most of the O2 evolution from H2O2 in the light. Our evidence indicates that this process: (1) is independent of the S states and insensitive to Tris washing, (2) turns over rapidly in high concentrations of peroxide, (3) yields 1 O2 per electron passing through system II; (4) dismutates two H2O2 molecules, so that there is no net consumption of 'holes'.


Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5'-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.


Studies on the mechanism of Tris-induced inactivation of oxygen evolution. A study was made of the inactivation by Tris of O2 evolution in chloroplasts and the subsequent reactivation of O2 evolution. We conclude: 1. At concentrations of Tris sufficient to inhibit O2 evolution directly, a slow rate (t 1/2 approximately 20--25 min) of inactivation occurs; 2. Inactivation is accelerated (t 1/2 approximately 2 min) by weak light absorbed by system II and is rate limited by a dark step with a half-time of about 200 s; 3. Minimally one quantum event within System II is sufficient to inactive 50--70% of the O2 evolving centers; 4. This process is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive but is inhibited by reduced dichlorophenol indophenol and phenazine methosulfate, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene and tetraphenylboron; 5. Partial reactivation of inactive O2 evolving centers is affected by the use of the same reagents inhibiting the light induced inactivations; 6. The life-time (t 1/2 approximately 1 to 3 h) of the activable state is correlated with diffusion across thylakoids of the larger manganese pool released from binding sites and remaining in thylakoids following inactivation of O2 evolution.


Damage to mitochondrial electron transport and energy coupling by visible light. The effect of treating mitochondria with visible light above 400 nm on electron transport and coupled reactions was examined. The temporal sequence of changes was: stimulation of respiration coupled to ATP synthesis, a decline in ATP synthesis, inactivation of respiration, increased ATPase activity and, later, loss of the membrane potential. Loss of respiration was principally due to inactivation of dehydrogenases. Of the components of dehydrogenase systems, flavins and quinones were most susceptible to illumination, the iron-sulfur centers were remarkably resistant to being damaged. Succinate dehydrogenase was inactivated before choline and NADH dehydrogenase. Redox reactions of cytochromes and cytochrome c oxidase activity were unaffected. Inactivation was O2-dependent and prevented by anaerobiosis or the presence of substrates for the dehydrogenases. Light in the range 400-500 nm was most effective and the presence of free flavins greatly enhanced inactivation of all of the above mitochondrial activities. This suggests that visible light mediates a flavin-photosensitized reaction that initiates damage involving participation of an activated species of oxygen in the damage propagation.


Control of respiration in proteoliposomes containing cytochrome aa3. II. Inhibition by carbon monoxide and azide. 1. Carbon monoxide (CO) acts competitively towards oxygen when the latter is taken up in respiration by cytochrome aa3-containing proteoliposomes, both in the presence of p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin (deenergized state) and in their absence (energized state). At high levels of CO, the double reciprocal plots (1/v vs. 1/[O2]) in the energized and deenergized states are parallel, i.e. energization acts "anti-competitively" towards oxygen, and the "respiratory control ratio" decreases as the oxygen concentration decreases. 2. Azide acts non-competitively towards cytochrome c when the latter is oxidized by cytochrome aa3-containing proteoliposomes both in the energized and deenergized (plus p-trifluoromethoxy carbonyl cyanide phenylhydrazone and valinomycin) conditions. At low azide concentrations the apparent Ki for azide is unaffected by energization, but at high azide levels the Ki increases in energized liposomes, i.e. the "respiratory control ratio" decreases as the azide concentration increases. 3. It is concluded that the inhibitor experiments are consistent with but do not prove the concept that the oxidase molecules in a single vesicle are responding to a single "energization state" or set of electrochemical gradients. This and other models are discussed.


Requirement of the light-harvesting pigment.protein complex for magnesium ion regulation of excitation energy distribution in chloroplasts. Cation regulation of excitation energy distribution was examined in chloroplasts isolated from (a) pea seedlings, grown in intermittent illumination, which contain no light-harvesting complex, (b) a barley mutant which is deficient in the major polypeptide component of the light-harvesting complex, and (c) a soybean mutant which contains a reduced amount of light-harvesting complex. It was found that: (1) Mg2+-induced increase in Photosystem II fluorescence at room temperature is small in the chloroplasts of the soybean mutant, smaller in the barley mutant, and almost absent in the light-harvesting complex-less chloroplasts of pea as compared to their respective controls. (2) Mg2+-induced increase in the F685/F730 emission peak ratio at 77 K is not detected in the isolated chloroplasts of the intermittent light-grown pea and the barley mutant. (3) Pre-illumination induced State 1-State 2 and adaptation in vivo is absent in the barley mutant and is less pronounced in the soybean mutant as compared to their respective controls. (4) Increase of slow fluorescence decay upon addition of Mg2+ observed in control chloroplasts was not detected in chloroplasts of intermittent-light grown peas. These results confirm earlier conclusions (Armond, P.A., Arntzen, C.J., Briantais, J.M. and Vernotte, C. (1976) Arch. Biochem. Biophys. 175, 54--63; Davis, D.J., Armond, P.A., Gross, E.L. and Arntzen, C.J. (1976) Arch. Biochem. Biophys. 175, 64--70) that light-harvesting complex is required for the Mg2+-induced regulation of the excitation energy distribution between Photosystems I and II. The characteristic P-S decay and I-D dip of the in vivo fluorescence inductions (Kautsky effect) were not significantly altered in the light-harvesting complex-less and the light-harvesting complex-deficient chloroplasts as compared to their respective controls. These results indicate that light-harvesting complex is not obligatorily required to observe the P-S decay or the I-D dip.


Induction patterns of delayed luminescence fromisolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield. 1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period. 2. Two distinct decay components of delayed luminescence were measured a "fast" component (from approximately 1 ms to approximately 6 ms) and a "slow" component (at approximately 6 ms). 3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change. 4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation. 5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics. 6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity. 7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant. 8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the "lake" model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the "w" hypothesis) were in closer agreement with the results.


Selective ion binding and membrane activity of synthetic cyclopeptides. Four cyclic peptides related to the membrane-active complexones PV, cyclo-(L-Pro-Lval-D-Pro-D-Val)3, and valinomycin were synthesized: (1) cyclo-(L-Pro-L-Ala-D-Val)3 or PVPA, (2) cyclo-(L-Ala-L-Val-D-Pro-D-Val)3 or PVAV, (3) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2-L-Pro-D-Val or PV-10, (4) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2 or PV-8. In a two-phase extraction assay the affinity of PV and PVPA for alkali picrates was about three orders or magnitude greater than that of valinomycin. It was about equal to valinomycin for PVAV and much lower for PV-10 and PV-8. PV, PVPA and PVAV showed a selectivity sequence similar to that of valinomycin, namely K+ approximately Rb+ greater than Cs+ greater than Na+ greater than Li+. In the series PV, PV-10, PV-, the preference for K+ over Na+ was 700, 5 and less than 1, respectively. Thus, it was possible to reverse the selectivity of PV for K+ over Na+ by reducing the ring size from 12 to 8 amino acid residues. In sheep red cell lipid bilayer membranes PVPA increased the membrane conductance significantly in the presence of either KCl or NaCl but it was less potent than PV. PV-10, PV-8 and PVAV on the other hand were ineffective in this assay. The inactivity of PVAV as a potassium carrier in membrane was in contrast to its high affinity for potassium picrate in two-phase assays. Such a behaviour may be observed of a compound that has too low an aqueous cation binding constant to use the solution-complexation mechanism of PV (Davis et al. (1976) Biochemistry 15, 768--774 and Pinkerton et al. (1969) Biochem. Biophys. Res. Commun. 35, 512--518) and too slow binding and release kinetics to use the interfacial-complexation mechanism of valinomycin.


Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells. Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin. Following supplementation with saturated fatty acids of longer than 15 carbons (100 micron) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth. Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35--41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72--82% after addition of unsaturated fatty acids. A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids. The results suggest that maintenance of membrane fluidity by unsaturated fatty acids in membrane phospholipids is critical to membrane integrity and cell growth.


Relation of cytolytic activity of an ether-deoxy lysophosphatidylcholine analog to available membrane surface. Comparison of normal and tumor cells from mice. 1-Hexadecylpropanediol-3-phosphorylcholine, an ether-deoxy analog of lysophosphatidylcholine, has been employed to study the sensitivity of various types of mouse cells with respect to changes in membrane permeability induced by lysophosphatidylcholine. Cells used included erythrocytes, thymocytes, spleen cells and macrophage, as well as 4 different tumors (2 lymphomas, 1 Ehrlich acites and 1 methylcholanthren-induced fibrosarcoma). The sensitivity to the lysophosphatide (on a per-cell basis) of the above cell types varied by a factor of 65. When lytic concentrations were related to available membrane surface, this variation was reduced to a factor of 2.5. No principal difference was observed between the sensitivity of normal versus tumor cell membranes with respect to lysophosphatidylcholine lysis. Membrane surface, available for lysophosphatidylcholine, has been estimated from binding equilibria of 14C-labelled deoxy-lysophosphatidylcholine to the cells under standardized conditions. This method is based on the finding that binding equilibria of lysophospholipids to cells are predominantly determined by the available membrane surface.


Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts. 1. Extracts of the human erythrocyte membrane have been prepared by solubilization with Triton X-100 and analysed by electrophoresis and gel filtration techniques. 2. The extracts have been incorporated asymmetrically into lecithincholesterol-n-decane planar bilayers. 3. The electrical characteristics and glucose permeabilities of the bilayers have been measured. 4. The extracts increased the electrical conductance of the bilayers and also markedly enhanced the D-glucose permeability but not the L-glucose permeability. 5. The enhanced D-glucose permeability was inhibited by monosaccharide transport inhibitors. 6. The results support the claim that a monosachharide facititated diffusion system has been set-up in vitro which has many of the characteristics of the transport system in the human erythrocyte membrane. 7. The data indicates that the trans membrane polypeptides of band 3 of the electrophoretogram of the erythrocyte membrane proteins (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2616) are implicated in D-glucose transport, although the possibility that relatively minor component of the membrane could be responsible for glucose transport cannot be eliminated.


Antibody-mediated targeting of liposomes. Binding to lymphocytes does not ensure incorporation of vesicle contents into the cells. Small sonicated lipid vesicles containing the water-souble fluorescent dye 6-carboxyfluorescein were formed from dioleoyl phosphatidylcholine and the antigenic lipid N-dinitrophenylaminocaproyl phosphatidylethanolamine. When these vesicles were incubated with trinitrophenyl-modified human lymphocytes and divalent anti-trinitrophenyl antibody, the antibody bound 5000 to 15 000 vesicles to each cell. Binding was detected by fluorescence microscopy and quantitated by fluorometry and flow microfluorometry. Binding was three times greater with F(ab')2 fragments than with the whole antibody and, as expected, was almost absent with the monovalent F(ab') fragments. It was also absent or greatly reduced, (i) with control immunoglobulin G, (ii) in the presence of excess soluble trintrophenyl hapten, or (iii) if hapten was omitted from either cells or vesicles. It was unaffected by sodium azide and 2-deoxy-D-glucose but was markedly decreased at 3 degrees C. It was not reversed by incubation at 3 degrees C with excess trinitrophenyl lysine. Self-quenching of the fluorescence of 6-carboxyfluorescein was used to distinguish between release of vesicle contents into the cells and simple binding of intact vesicles (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Gagins, W.A. (1977) Science 195, 489--491). Antibody-mediated binding led to little or no increase over spontaneous background levels in the amount of vesicle contents released into the lymphocytes.


Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates. The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.


A simple apparatus for performing short-time (1--2 seconds) uptake measurements in small volumes; its application to D-glucose transport studies in brush border vesicles from rabbit jejunum and ileum. An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10--20 microliter. Using this technique the kinetic parameters Km and V of D-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the Km for D-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.


Differential effects of erythrocyte lysates on spectra of potential-sensing carbocyanine dyes. A recent report by Hladky, S.B. and Rink, T.J. ((1976) J. Physiol. 263, 287--319) demonstrates the binding of a thiocarbocyanine dye (di-S-C3 (5)) to an intracellular constituent from human erythrocytes. Evidence presented below shows that the binding of carbocyanine dyes depends on the particular type of dye used and on the species of erythrocyte from which cellular constituents are prepared. It is recommended that dyes which show minimal binding to intracellular components be used for investigations where carbocyanine dyes are employed as probes of membrane potential.


Amiloride-induced stimulation of HCO-3 reabsorption in turtle bladder. Amiloride in the mucosal fluid (at concentrations of 5 . 10(-6) M to 10(-4) M) reversibly stimulates the HCO-3-dependent moiety of the short-circuiting current (Isc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl-. This effect is uniquely different from the known inhibitory effect of this agent on Na+ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response range should take into account its effect on HCO-3 transport.


Neurite formation and membrane changes of mouse neurobalstoma cells induced by valinomycin. A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K+ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.


A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation. Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.


Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles. The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.


Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor. Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.


Mapping of deoxydi- and trinucleotides liberated by the action of endonucleases from the silkworm and Aspergillis oryzae. Both silkworm nuclease and nuclease O of Aspergillus oryzae mycelia hydrolyse DNA endolytically to di- and trinucleotides terminating in 5'-phosphate. These oligonucleotides were fractionated first be DEAE-cellulose chromatography with 7 M urea into the respective isoplithic groups and then analysed for the composition and isomerism: each group was labeled 5'-terminally by the [gamma-32P]ATP-polynucleotide kinase reaction and then electrophoresed monodimensionally for the dinucleotides and two-dimensionally for the di- and trinucleotides mixtures, respectively, followed by elution and digestion with snake venom exonuclease. Both nucleases gave rather similar simple maps in which all sixteen dinucleotides and almost all the possible trinucleotides were identified, indicating their random mode of actions.


Non-coordinated histone synthesis with DNA replication and close temporal association of ribosomal proteins and rRNA syntheses in germinating Vicia faba embryos. Syntheses of DNA, RNA and basic proteins during germination of Vicia faba seeds were examined. Incorporation of [3H]thymidine into DNA reached a maximum peak at approximately 34 h after beginning imbibition and the phase of DNA replication was preceded by active RNA synthesis. When Vicia embryos were labeled with [3H]lysine or E13H]arginine, these isotopes were incorporated into ribosomal proteins and histones. The incorporation studies indicated that the synthesis of ribosomal structural proteins is closely linked with rRNA synthesis and histone synthesis occurs during and also before the phase of DNA replication. Polyacrylamide gel electrophoresis of Vica histones indicated that some histones of Vicia embryos differ from corresponding calf thymus histones in their mobilities.


Cleavage of type 2 adenovirus DNA by HaeIII endonuclease. I. Catalog of HaeIII fragments. Tye 2 adenovirus DNA was divided into 14 fragments by sequential use of BamI, HsuI, SmaI, anc EcoRI endonuclease. Each fragment was purified by gel electrophoresis and subsequently cleaved with HaeIII endonuclease. From the number of fragments produced, we could calculate the number of HaeIII cleavage sites: there are a total of 187 sites. HaeIII sites were not randomly distributed along the adenovirus chromosome. Most sites were clustered in the G + C-rich left half of the chromosome. The sum of the molecular weights of the HaeIII fragments is 22.4 . 10(6), within 2 % of the molecular weight of adenovirus DNA (22.9 . 10(6).


Cleavage of type 2 adenovirus DNA BY HaeIII endonuclease. II. Map of HaeIII sites in EcoRI fragments C and E. Fourteen HaeIII cleavage sites have been located in EcoRI fragments C and E which together constitute the rightmost 16.6% of the type 2 adenovirus chromosome. In addition, nine sites recognized by SmaI, BalI, HsuI, HpaI, and HpaII endonucleases have also been mapped in this region.


Base composition studies on transfer RNA from normal and regenerating rat liver. The base composition of bulk tRNA isolated from regenerating rat liver, 12, 18, 24 and 30 h after partial hepatectomy, was determined by a 3H derivative method. Only a few minor statistically significant changes (2--11%), as compared to sham-operated liver, were found at 18, 24 and 30 h after hepatectomy. These included a reduction in the amounts of adenosine and 3-(3-amino-3-carboxypropyl)-uridine, and an increase in the amounts of 1-methyl-adenosine, 1-methylguanosine, 3-methylcytidine and pseudouridine. Similarly, when the base composition of tRNA fractions from control and 24-h regenerating rat liver, partially purified by one-dimensional polyacrylamide gel electrophoresis, was determined, no gross differences were observed. These results suggest that the process of liver regeneration is not accompanied by a gross alteration of the modification pattern of tRNA.


Uncoupling of amino acid turnover on transfer RNA from protein synthesis in HeLa cells. The aminoacylation of tRNA has been investigated in relation to protein aynthesis in living HeLa cells. In cells growing normally, the rates of tRNA charing are compatible with the observed entry of amino acids into protein. In contrast, when protein synthesis is inhibited 95--98% by either reduced temperature or cycloheximide, aminoacylation of tRNA is relatively unaffected. We conclude that, under these conditions, the aminoacylation of tRNA is uncoupled from subsequent steps in protein synthesis. These results provide for the first time a possible biological role for the observed aminoacyl-tRNA hydrolase activities of the tRNA synthetases.


Amino acid incorporation in a cell-free system derived from rat liver studied with the aid of selenodiglutathione. Selenodiglutathione (GSSeSG), a potent inhibitor of elongation factor 2 (EF2) has been used to study amino acid incorporation in a rat liver cell-free system. While translocation of the ribosomes was inhibited by GSSeSG, ribosomes with a free acceptor site were still capable of incorporating one amino acid residue. From this the average number of amino acids incorporated per ribosomes was calculated to be 2--5. In this respect virtually no difference has been observed between ribosomes present on small or large aggregates. The time required for one translocation by all active ribosomes, and the time required for the incorporation of one amino acid (starting with aminoacyl-tRNA or amino acids) has also been determined. By incubation under conditions for amino acid incorporation, part of the ribosomes were completely inactivated whereas the rest remained as active as at the start of the incubation.


Template activity for histones of poly(A)-minus RNA fraction from different developmental stages of Artemia salina embryos. Template activity for histones of RNA fractions derived from Artemia salina embryos at different developmental stages were measured in a Krebs ascites cell-free system. Appreciable amounts of acid-soluble polypeptides comigrating with Hela cell histone markers on acrylamide gel electrophoresis were detected only when RNA fractions from nauplii were used. Tryptic peptide analysis by high voltage electrophoresis of the translational products had a pattern qualitatively similar to that of in vivo labeled histone markers from Hela.


The properties of hydrogenase from Thiocapsa roseopersicina. The soluble and chromatophore-bound hydrogenases from the purple sulphur bacterium Thiocapsa roseopersicina strain BBS were purified up to homogeneity and the properties studied. The amino acid composition of hydrogenase preparations from different fractions of T. roseopersicina is identical, glycine and alanine as N-terminal amino acid residues. In comparison with other hydrogenases, especially in the immobilized state, the preparations obtained are shown to be more stable to O2 during storage and they are characterized by high thermal stability. Inactivation is observed above 78--80 degrees C and the optimal temperature for enzyme action is 70 degrees C. The homogeneous enzyme preparations catalyse the exchange reaction between 2H2 and H2O and reversible redox reactions of methyl viologen and benzyl viologen as well as H2 formation from reduced ferredoxin. According to our data, the hydrogenase of T. roseopersicina bound with chromatophores is identical to the soluble one.


The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes. The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.


An essential active-site histidine residue in human prostatic acid phosphatase. Ethoxyformylation by diethyl pyrocarbonate and phosphorylation by a substrate. Human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) is a dimeric (alpha 2) protein that catalyses the hydrolysis of phosphomonoesters. Several reports suggest that a phosphoenzyme intermediate is involved in the mechanism of acid phosphatase. Chemical modification studies and trapping experiments were therefore undertaken in order to ascertain the identity of the amino acid residue(s) involved in the formation of this intermediate. Human prostatic acid phosphatase is inactivated by diethyl pyrocarbonate (second-order rate constant of 7 M-1. min-1 at pH 6.2) with an accompanying increase in absorbance at 242 nm due to formation of ethoxyformylhistidyl derivatives. In the presence of competive inhibitors the rate of inactivation is decreased. Inactivation can be partially reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue having a pK alpha of 6.5. Direct evidence for the involvement of a histidine residue in the mechanism was obtained by trapping a covalent phosphohistidyl-enzyme intermediate. Incubation of the enzyme with p-nitrophenyl [32 P] phosphate leads to incorporation of 0.44 mol 32P/mol enzyme. The denatured phosphoenzyme,which was acid labile but base stable, was hydrolyzed in 3 M KOH and the radioactivity was found to cochromatograph with synthetic tau-phosphohistidine on Dowex-1 ion-exchange resin. These results are consistent with a catalytic mechanism involving histidine as a nucleophile in the formation of a covalents phosphoenzyme intermediate.


An essential arginine residue in human prostatic acid phosphatase. Treatment of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with either of the arginine-specific modifiers 2,3-butanedione or 1,2-cyclohexanedione in borate buffer at pH 8.1 leads to loss of activity. The inactivation by cyclohexanedione can be partially reversed by 0.2 M hydroxylamine. The rate of inactivation by both modifiers is decreased in the presence of the competitive inhibitors L-(+)-tartrate or inorganic phosphate but not in the presence of the non-inhibitor D-(-)-tartrate. Amino acid analysis of modified acid phosphatase indicates that only arginines are modified and that L-(+)-tartrate protects at least two arginyl residues from modification. A likely role of these arginyl residues is their involvement in binding the negatively charged phosphate group of the substrate.


The multiple forms and kinetic differences of rat colonic beta-N-acetylhexosaminidases. Rat colonic beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been separated into three forms by DEAE-cellulose chromatography with an increasing salt gradient. It was not possible to separate the glucosaminidase activity from the galactosaminidase activity by a variety of chromatographic procedues, but the ratio of the two specific activities varied during purification. The pH optima were however identical, for both activities and all three forms. Kinetic measurements including inhibition by substrate analogues showed differences between the two activities as well as among the three forms. A common active site model was inconsistent with the results. Data from mixed substrate experiments were consistent with a model wherein the two activities reside in seperate active sites, each able to be inhibited by the substrate for the other site. The effect of acetate and SH reagents confirmed the two-site model. Treatment with neuraminidase, thimerosal, p-hydroxymercuribenzoate, HgCl2 and AgNO3 or heating at 50 degrees C did not produce any effect on the A form that could be identified as a conversion to the B form. Measurement of the effects on both activities supported the two-site model. It is concluded that the relationship between the A and B forms in the rat colonic mucosa hexosaminidases must be different from that reported for such enzymes from other sources.


In vitro prothrombin synthesis from a purified precursor protein. II. Partial purification of bovine carboxylase. In this paper, we describe the isolation and partial purification of an enzyme system that converts bovine decarboxyfactor II (PIVKA-II) into prothrombin (factor II). It is shown that the increase in factor II activity occurs in parallel with 14CO2 incorporation into BaSO4 adsorbable proteins. The system is not strictly vitamin K-dependent because it is obtained from the livers of normal healthy cows. By preincubating the enzyme(s) with an excess of warfarin, an absolute vitamin K1-dependence can be obtained. The reaction is inhibited by its own product, factor II.


Enzymatic oxidation of mercury vapor by erythrocytes. The formation of glutathione radicals, the evolution of nascent oxygen or the peroxidatic reaction with catalase complex I are considered as possible mechanisms for the oxidation of mercury vapor by red blood cells. To select among these, the uptake of atomic mercury by erythrocytes from different species was studied and related to their various activities of catalase (hydrogenperoxide : hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and glutathione peroxidase (glutathione : hydrogen-peroxide oxidoreductase, EC 1.11.1.9). A slow and continuous infusion of diluted H2O2 was used to maintain steady concentrations of complex I. 1% red cell supsensions were found most suitable showing high rates of Hg uptake and yielding still enough cells for subsequent determinations. The results indicate that the oxidation of mercury depends upon the H2O2-generation rate and upon the specific acticity of red-cell catalase. The oxidation occurred in a range of the catalase-H2O2 reaction where the evolution of oxygen could be excluded. Compounds reacting with complex I were shown to be effective inhibitors of the mercury uptake. GSH-peroxidase did not participate in the oxidation but rather, was found to inhibit it by competing with catalase for hydrogen peroxide. These findings support the view that elemental mercury is oxidized in erythrocytes by a peroxidatic reaction with complex I only.


Physical properties and Tris inhibition of an insect trehalase and a thermodynamic approach to the nature of its active site. The midgut from Rhynchosciara americana larvae display a trehalase (alpha,alpha'-trehalose glucohydrolase, EC 3.2.1.28) which is soluble with a molecular weight of 122 000 and pI 4.6. The optimum pH of the enzyme is 6.0, its apparent Km for trehalose is 0.67 mM and its energy of activation is 16.7 kcal/mol. Sulfhydryl reagents do not inhibit the trehalase. The results suggest the existence of two carboxyl groups in the active site, one of which has a very high (8.3) pK. The increase of the pK values of the essential groups of the free enzyme in the presence of increasing concentrations of dioxane supports the hypothesis that these groups are carboxyls. The purified enzyme hydrolyzes only alpha,alpha'-trehalose and it is competitively inhibited by several compounds.


The interaction of a trypsin-dependent neutral protease and its inhibitor found in tumour cells. Analysis of complex kinetic data involved in a thiol-disulphide exchange mechanism. Ehrlich ascites tumour cells contain a granule-derived zymogen which on trypsin activation yields a collegenolytic neutral protease. The preparation of the granule fraction by subcellular fractionation procedure results in the preparation of a second fraction referred to as the post-granule supernatant fraction. The post-granule supernatant fraction contains a latent form of the granule-derived neutral protease and an excess of cytoplasmic inhibitor for this enzyme. The inhibitor of neutral protease is also capable of inhibiting trypsin and in each case the chemical mechanism of enzyme.inhibitor complex formation has been shown to be a reversible thiol-disulphide exchange. The post-granule supernatant fraction exhibited complex kinetic data when the interactions between the inhibitor, the latent enzymes and trypsin were examined simultaneously by incremental analysis. The data were interpreted and quantitatively analysed by computer analysis. It was demonstrated that the conventional types of analysis could not have provided meaningful interpretations of the experimental data provided by these complex-interacting systems.


Separation of human renal renin and pseudorenin by affinity chromatography on hemoglobin-Sepharose-2B. Human renal renin (EC 3.4.99.19) and pseudorenin were easily separated in a single step by affinity chromatography on hemoglobin-Sepharose-2B. Renin and pseudorenin were monitored by their actions on crude and partially purified hog protein renin substrates at neutral and acidic pH and on synthetic labelled polymeric renin substrate. Under the conditions employed (0.1 M sodium acetate (pH 3.5)/1 M sodium chloride at 4 degrees C) renin does not bind to the affinity adsorbent while pseudorenin is effectively bound and can be eluted only after raising the pH to 6.5. Pseudorenin-free renin prepared by this method is devoid of proteolytic activity toward hemoglobin. The chromatographic behaviour of renal pseudorenin on hemoglobin-Sepharose-2B is similar to that of cathepsin D.


Purification and kinetic properties of human erythrocyte Mg2+-dependent inorganic pyrophosphatase. Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte hemolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 mumol PPi hydrolysed.min-1.mg-1 at pH 8 and 37 degrees C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg2+ concentration in different ways. This ion constitutes part of the substrate (the Mg.PPi complex; Km = 1.4.10(-4) M) and probably acts as an allosteric activator (kinetic activation constant: KMg2+a = 7.5.10(-4) M). Equilibrium binding studies performed in the absence of PPi showed 4 binding sites for Mg2+, all having the same high affinity (dissociation constant: KMg2+d = 4.10(-6) M). Since the concentration of free Mg2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.


Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo. 1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.


A specific and reversible macromolecular inhibitor of phenylalanine ammonia-lyase and cinnamic acid-4-hydroxylase in gherkins. A non-dialysable inhibitor of phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, EC 4.3.1.5) has been partially purified from dark-grown gherkin hypocotyls. On extraction of tissue it is found both in the soluble (106 000 X g supernatant) and microsomal (106 000 X g pellet) fractions and can be extracted from the microsomal membranes with 10 mM sodium cholate and 1 M KCl. The soluble and microsomal fractions have similar properties, suggesting the presence of the same active component. The inhibitor is small (Mr less than 20 000), thermolabile, sensitive to proteolytic digestion, and apparently hydrophobic. Purification of the inhibitor was achieved by chromatography on DEAE-cellulose by gel filtration on Sephadex G-50. The inhibitor preparations inhibit phenylalanine ammonia-lyase isolated from a number of plant tissues and also cinnamic acid-4-hydroxylase (trans-cinnamate, NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11) from gherkins and peas, but not a wide range of other enzymes. The evidence suggests that inhibition of the two enzymes is due to the same substance, but this has not yet been confirmed. Kinetic experiments show that the inhibitor is competitive with phenylalanine for the lyase and that its association with the lyase is reversible. Further, a mixture of inhibitor and lyase can be separated on non-denaturing polyacrylamide gels without loss of lyase activity. The activities of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase are often concurrently regulated and both have regulatory roles in phenol metabolism; it is suggested that the inhibitor may be specifically involved in controlling their activities in vivo.


Purification and properties of two succinate semialdehyde dehydrogenases from human brain. In human brain there are two major isoenzymes of succinate semialdehyde dehydrogenase (succinate-semialdehyde: NAD+ oxidoreductase, EC 1.2.1.24). They are composed of two apparently identical subunits with a molecular weight of 69 000. The Km (limits) for their substrates NAD+ and succinate semialdehyde are 1.6.10(-5) M and 3.7.10(-6) M, respectively, for one enzyme, and 1.85.10(-5) M and 2.10(-6) M, respectively, for the other. These values, and other kinetic data obtained from the two enzymes are not very different. However the enzymes differ in the following respects: their behavior on ion exchange and 5'-AMP affinity columns, their isoelectric points, their tryptic fingerprints and in their amino acid compositions.


Serine transhydroxymethylase. Equilibrium binding of folate analogs as active site probes. Formation of a quinoid-like structure within the glycyl-pyridoxal phosphate moiety of serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyltransferase, EC 2.1.2.1) is dependent upon the dissociation of the 2-S hydrogen of glycine which in turn requires the presence of tetrahydrofolate or analogs thereof. Equilibrium binding studies with the series folate, dihydrofolate, and tetrahydrofolate showed that reduction of the pteridine ring enhances both quinoid formation and binding. A 5,8-deazafolate series showed that modifications in the 4 position, 10 position and the glutamyl position yield interrelated alterations of quinoid formation which could not be correlated with binding.


Evidence for significant quantities of creatine kinase MM isoenzyme in human brain. The isoenzymes of creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) in human brain were studied. Approx. 30% of total creatine kinase activity is due to the MM isoenzyme. The identity of this isoenzyme was confirmed by several techniques and shown not to be of mitochondrial origin.


Chromatographic study on the specificity of bis-p-nitrophenylphosphate in vivo. Identification of labelled proteins of rat liver after intravenous injection of bis-p-nitro[14C]phenylphosphate as carboxylesterases and amidases. The procedure established to isolate the carboxylesterases E1, E2 and EA from rat liver (Arndt, R. and Krisch, K. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 589-598) was applied to characterize in vivo bis-p-nitro[14C]-phenyl-P-labelled proteins. The peaks of radioactivity and of residual enzyme activities (hydrolysing methylbutyrate, p-nitrophenylacetate and acetanilide) were found in the same peaks after column chromatography and could be related to the well-defined esterases E1, E2 and EA. There is no indication of a nonspecific binding of bis-p-nitrophenyl-P or of one of its metabolites. The relative quantitative amounts of E1, E2 and EA were calculated to represent 40, 14 and 46%, respectively, of the total carboxylesterase content of rat liver. The relative amount of bound (not dialysable) radioactivity in rat liver depended on the survival time. During purification, the yield of enzyme activities corresponded to that of bound radioactivity, confirming the specificity of bis-p-nitrophenyl-P in vivo. Hence the radioactive metabolites of the inhibitor obviously do not possess binding affinities of quantitative importance to the rat liver proteins.


Hepatic fatty acid oxidation and ketogenesis after clofibrate treatment. The effect of clofibrate treatment on hepatic ketogenic capacity was studied in rats. Ketogenesis from octanoate and oleate was increased 2- and 4,5-fold, respectively, in hepatocytes from fed, treated rats. In contrast to controls ketogenic rates did not increase upon starvation. While ketogenesis from oleate was higher in fed, treated animals than in fasted controls, endogenous ketogenesis was lower and increased upon starvation. Ketogenesis from octanoate and oleate was stimulated approx. 2-fold in homogenates from treated animals. Labeled pyruvate and succinate oxidation was unaltered. [1-14C]Oleate oxidation was severely inhibited by cyanide, both in homogenates from controls and treated animals. Clofibrate caused a 3-fold increase in hepatic carnitine levels. Catalase and glutamate dehydrogenase activities were also increased by the drug. Cytochrome c oxidase did not change. Despite their increased ketogenic capacity hepatocytes from treated rats esterified as much oleate as controls. The increased oxidation was matched by an increased oleate uptake. Plasma ketones were increased 2-fold in fasted, treated animals. Plasma free fatty acids were unaffected. It is concluded that the enhanced ketogenic capacity induced by clofibrate is the result of an increase in mitochondrial beta-oxidation, an increase in the activity of carnitine palmitoyltransferase and possibly of the observed increases in hepatic carnitine content and fatty acid uptake.


Bile acid pool changes and regulation of cholate synthesis in experimental diabetes. The effect of alloxan-diabetes and insulin treatment in bile acid pool size and composition, bile acid secretion and cholic acid synthesis was investigated in the rat. The size of the cholate pool was significantly increased 4 days after diabetes induction. It reached a constant size three times that of control animals after 2 weeks of diabetes. Changes in bile acid pool size and secretion were directly dependent of the insulin deficiency state since they were reversed by insulin treatment and were not influenced by the caloric intake of the animal nor the pharmacologic effect of alloxan. Biliary cholate secretion was also 3-fold increased in diabetic rats and it accounted for more than 80% of the total bile acids compared to 60% in the control group. The calculated daily rate of cholate synthesis was increased in diabetic rats and the circadian rhythm of cholate synthesis was abolished in this condition. Therefore, it was shown that the negative feedback mechanism that regulates bile acid snythesis was deleted in diabetes. This mechanism was partially restored after 2 weeks of insulin treatment. These studies demonstrated that bile acid metabolism was profoundly changed in alloxan-diabetic rats and suggested that insulin may play an important role in the regulation of bile acid snythesis and intestinal absorption.


15-Hdroxyprostaglandin dehydrogenase activity in vitro in lung and kidney of essential fatty acid-deficient rats. Weanling rats were fed for 6 months on a diet deficient in essential fatty acids: either fat-free, or with 28% (w/w) partially hydrogenated fish oil. Control rats were fed a diet with 28% (w/w) arachis oil for 6 months. 15-Hydroxyprostaglandin dehydrogenase activity was determined as initial rates of formation of 3H-labelled 15-keto-dihydro-prostaglandin E1 plus 15-keto-prostaglandin E1 in high speed supernatants of lung and kidney from each of the groups of rats. Dehydrogenase activity (expressed as either pmol/min per mg soluble protein, or as nmol/min per g tissue) was decreased 30--40% in the lungs of the two groups on diets deficient in essential fatty acids as compared to the control group. No difference were observed in dehydrogenase activity in the kidneys. The dehydrogenase may be of importance for the regulation of the level of endogenous prostaglandins and, thus, a decrease in activity could result in a slower turnover of prostaglandins.


Localization of cytochrome P-450-linked mixed function oxidase and steroid hydroxylations within zones of bovine adrenocortices. The localization of contents and activities of components of cytochrome P-450-linked mixed function oxidase system and the hydroxylation of steroids in mitochondria and microsomes within zones of adrenocortices was investigated. NADPH-adrenodoxin reductase activity was higher in the zona glomerulosa than in the other zones. Adrenodoxin content was present at high concentration in the zona fasciculata. The localization of cytochrome P-450 within zones of adrenocortices has already been reported (Ichikawa, Y., Kuroda, M. and Yamano, T. (1970) J. Cell Biol 45, 640--653). The zona fasciculata contained more mitochondrial and microsomal cytochromes P-450 than the zona glomerulosa or zona reticularis. Cleavage activity of the side chain of cholesterol was found in three zones. However, particularly high activity was observed in the zona fasciculata and zona reticularis. 11beta-Hydroxylation was found in the zona glomerulosa. 21-Hydroxylation activity was especially higher in the zona fasciculata than in the other zones.


Enzymic hydrolysis of diol lipids by pancreatic lipase. Chain length and positional specificities of pancreatic lipase for diol lipids have been examined by use of the synthetic substrates such as the diol esters and related esters that contain C6--C20 even-numbered saturated acids and oleic acid as fatty acids, and ethylene glycol, 1,2- and 1,3-propanediols, 1,3-, 1,4-, and 2,3-butanediols, monohydric alcohols, and glycerol as alcohols. No remarkable difference in the degree of hydrolysis was observed in the case of diol esters having C14--C20 fatty acids at least during 30-min digestion, though hydrolysis reached a maximum in diol diotanoates after the same period. Pancreatic lipase specifically released only the fatty acids esterified with the primary hydroxyl groups of diols.


Lipid adaptation in liver mitochondrial membranes of carp acclimated to different environmental temperatures: phospholipid composition, fatty acid pattern and cholesterol content. The lipid fraction of liver mitochondria has been studied in carp acclimated to high and low environmental temperatures. Evidence is provided for a temperature-induced lipid adaptation which might control membrane fluidity. This supports suggestions made in a recent communication on temperature-induced changes in the Arrhenius functions of mitochondrial oxidase systems from carp liver ((Wodtke, E. (1976) J. Comp. Physiol. 110, 145--157). The results of the analysis of lipid composition are: the ratio of phospholipid to protein does not differ at cold and warm acclimation temperatures. Fish kept at low environmental temperature show a decreased molar ratio of cholesterol : phospholipid in mitochondrial membrane lipids; the diminished complexation renders phospholipids more sensitive to fluidity control by fatty acid substitution. A decrease of mitochondrial phosphatidylcholine at low acclimation temperature is observed, which is compensated by increased amounts of phosphatidylethanolamine and phosphatidylinositol. This means there is an increase in the acidic character of the phospholipids at low environmental temperature, and might be the cause of fluidization of the membrane and a decrease in transition temperature. The fatty acid pattern of carp mitochondria differs markedly from that of mammalian mitochondria; it is not identical for total lipids, phosphatidylcholine and phosphatidylethanolamine, and is roughly characterized by high amounts in n--3, but low amounts in n--6 and mono-unsaturated fatty acids. Low environmental temperature decreases the proportion of saturated species and markedly lowers the ratio of the alpha-linolenic acid family, the latter perhaps being caused by the increased activity of delta6-desaturase, as calculated for low acclimation temperature. An increase in mean unsaturation and in the proportion of 1,2-diunsaturated phospholipids most probably increases membrane fluidity at low acclimation temperature.


Effect of a choline inhibitor (N-isopropylethanolamine) on cellular metabolism of L-M cells. N-Isopropylethanolamine, a choline analog, is incorporated into L-M cell lipids as 1,2-diacyl-sn-glycero-3-phosphoisopropylethanolamine de novo and not by base exchange. In addition, the N-isopropylethanolamine effectively blocks choline uptake, which is a reversible process. The following specific time-dependent changes in cell metabolism also occur when N-isopropylethanolamine is present: (1) a decrease in total content of phosphatidylcholine, (2) inhibition of both the cellular uptake of [3H]choline and its incorporation into phosphatidycholine, (3) a decrease in the incorporation of [3H]thymidine into DNA as early as 2 h after initiating the N-isopropylethanolamine block, (4) inhibition of the cellular uptake of [3H]uridine and incorporation into RNA 16--24 h after addition of the N-isopropylethanolamine, and (5) stimulation of the cellular uptake of [3H]leucine and an inhibition of its incorporation into protein, which reached a maximum (68% of controls) 8 h after N-isopropylethanolamine treatment.


A spectral study of human ceruloplasmin. The absorption, luminescence and CD spectra of human ceruloplasmin were studied. The absorption spectrum in the infrared, and the CD spectrum in the ultraviolet, both indicate the presence of beta conformation in the native structure of the protein. From the magnitude of the measured ellipticity, it is estimated that 0.46 of the amino acid residues are in the beta conformation, the remaining 0.54 being in unordered form. A comparison of the fluorescence and phosphorescence properties of native and apoceruloplasmin shows that the presence of copper causes the quenching of tryptophanyl luminescence, probably through energy transfer to the copper chromophores. By the combined resolution of the absorption and CD spectra, it was concluded that the copper chromophores are involved in six electronic transitions in the region 300--900 nm. Our results provide evidence for an interaction between the copper chromophores responsible for the 330 nm absorption in ceruloplasmin.


Relationship of thermal quenching of protein fluorescence to intramolecular structural mobility. Temperature dependence (over the range from 5 to 75 degrees C) of the fluorescence quantum yield--the parameter sensitive to the rapid (ns) mobility--has been investigated. For native proteins containing 1--2 fluorescing chromophores this dependence in the non-denaturing temperature range can be described by the equation 1/q = a + b . T/eta (a and b are temperature-independent constants, T is the temperature (K), eta is the viscosity of water, (cP). The linearity of the plot of 1/q vs. T/eta probably indicates that the internal mobility of different parts of the protein globule is limited by the diffusion of the molecules of the solvent. The peculiarity of individual protein structures is reflected by various absolute values of the constants of quenching rates. The results of the studies indicate that an analysis of the temperature dependence of the protein fluorescence quantum yield (thermal quenching curves) might be a new experimental approach to the study of the mobility of protein structures. The thermal quenching curves of proteins could be used as a measure of the temperature dependence of the rapid reverse structural mobility.


Heme sulfuric anhydrides as soybean leghemoglobin structure probes. Mesoheme monosulfuric anhydride reacts at three distinct sites in soybean apoleghemoglobin a, at lysine-6, lysine-19 and lysine-57, the last one being the major site of reaction. The heme peptides obtained from thermolytic and pronase hydrolysates of the anhydride-leghemoglobin a were purified and correlated with the known amino acid sequence of the protein. Mesoheme bissulfuric anhydride also reacts with soybean apoleghemoglobin a giving a complex mixture of hemepeptides after hydrolysis with pronase. The visible spectrum of anhydride leghemoglobin is that of low spin heme. This suggests that anhydride leghemoglobin has a conformation with a covalent attachment via propionic acid side chain to lysine-57 and the sixth coordination position of the heme iron occupied by the distal histidine at position 61. Native leghemoglobin is assumed to exist in a similar type of configuration at low temperature, but with the heme propionate side chain being involved in a salt bridge with lysine-57.


Amino acid sequence of chicken fibrinopeptide A. Chicken fibrinopeptide A was isolated and the complete amino acid sequence was established. It is a pentadecapeptide with the sequence of Gln(Pyr)-Asp-Gly-Lys-Thr-Thr-Phe-Glu-Lys-Glu-Gly-Gly-Gly-Gly-Arg and is homologous with mammalian fibrinopeptide A. Two peptides which appear to be derivatives of fibrinopeptide A were also isolated. One of these appeared to be fibrinopeptide A with NH2-terminal pyroglutamic acid; the other was fibrinopeptide A which lacked the COOH-terminal arginine residue.


The increase in human antithrombin III tryptophan fluorescence produced by heparin. The increase in fluorescence of human antithrombin III has been used to study the binding of a semi-synthetic heparin analogue. One molecule of heparin were found to bind with an association constant of 8.9 . 10(4) M-1. The intrinsic fluorescence of antithrombin III exhibits a fluorescence quantum yield of 0.17. Upon addition of heparin a marked increase in the protein fluorescence quantum yield is observed.


The breakdown of spectrin produced by ultrasonication. The application of carefully controlled ultrasonication to human erythrocyte membranes (ghosts) has been found to produce a selective breakdown of the two spectrin polypeptides. This breakdown increases with the time and the intensity of ultrasonication. The 240 000 molecular weight spectrin polypeptide is slightly more susceptible to ultrasonication than the 220 000 molecular weight polypeptide. Isolated spectrin behaves in an identical manner when ultrasonicated. When highly purified samples of spectrin are ultrasonicated the progressive reduction of the molecular weight of the products with increasing time is clearly apparent.


Electrophoretic isolation and partial characterization of a major secretory glycoprotein from the submandibular glands of the mouse. After either cholinergic or adrenergic stimulation of the submandibular glands of the mouse, a major protein of the incubation medium could be isolated by electrophoresis, designated the AM2 protein. About 5 per cent of the secreted proteins and 2.4 per cent of the secreted protein-bound sialic acid was recovered as the purified AM2 protein. The AM2 protein appeared to be electrophoretically pure in 7.5% polyacrylamide gel both at pH 8.9 and at pH 4.3. In sodium dodecyl sulfate-electrophoresis the molecular weight was estimated to be about 80 000 for the major component and about 40 000 for the minor component. By isoelectric focusing the isoelectric point has been determined to be 4.7. The amino acid analysis indicated Glx, Asx, Leu and Ala as the major amino acids, comprising 15.0, 10.6, 9.2 and 9.1 per cent of the amino acid residues, respectively. The ratio of the acidic amino acids and their amides (Glx plus Asx) to the basic amino acids (Lys plus Arg) was 2.2. The sugar analysis showed that the AM2 glycoprotein consists of 17.3 per cent of carbohydrate, with as major carbohydrate component glucosamine. The molar ratio of the sugars was Man : Gal : Glc : GlcNH2 : sialic acid = 2.3 : 1.0 : 4.7 : 9.8 : 2.9. Galactosamine could be detected as a trace component and fucose was not detectable.


Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns. Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.


Isolation of purified insoluble aortic collagen. The chemistry of collagen from major blood vessels, such as the aorta, is poorly defined because of problems encountered in solubilization techniques. Normal extraction of calf aorta with acetic acid and/or pepsin does not yield significant quantities of collagen. However, treatment of the aorta with purified pancreatic elastase results in a residue containing a significant portion of the collagen. The amino acid analysis, the acrylamide gel electrophoretic patterns and the electron micrographs of this residue display characteristics consistent with relatively pure collagen. Using this purified collagen preparation approximately 90% of the collagenous material present can be solubilized by pepsin treatment.


The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen. Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.


Blue-fluorescent bovine alpha-crystallin. Blue-fluorescent alpha-crystallin has been isolated from bovine lenses by gel-filtration on Sephacryl S-200 Superfine. The blue fluorescence of this alpha-crystallin is characterized by fluorescence peaks at about 410 and 435 nm and two excitation peaks at about 350 and 370 nm. This finding suggests the existence of two different blue-fluorescences in bovine alpha-crystallin. Both low molecular weight alpha-crystallin and higher molecular weight alpha-crystallin exhibit similar blue fluorescence. With aging, in the nuclear region of bovine lenses, blue fluorescent low molecular weight alpha-crystallin shifts to non-covalently-linked higher molecular weight aggregates which are also blue-fluorescent.


Quantitation of epinephrine- and glucagon-sensitive adenylate cyclases of rat liver. Implications of alterations of enzymatic activities during preparation of particulate fractions and membranes. Stimulated and basal adenylate cyclase activities from livers of young and old rats were lower in particulates than in homogenates. Particulates were compared to homogenates by reconstituting the suspensions to the volume of the homogenates from which they were derived; enzyme activities in paired homogenates and particulates therefore reflected the same amounts of membrane-bound enzyme. The magnitude of the losses of hormone-sensitive activities in particulates was dependent on the age and sex of the animals and the concentrations of hormone. Particulates from 3-month-old animals showed glucagon-(1 . 10(-5) M) and epinephrine-sensitive (1 . 10(-4) M) activities which were 67 and 78% of homogenate activities, respectively; particulates from 24-month-old animals had activities relative to homogenates of 55% for glucagon and as low as 32% for epinephrine. The glucagon dose vs. response curve in particulates and membranes showed maximal activity at 1 . 10(-7) M glucagon while in homogenates activity increased linearly with increasing glucagon concentrations up to 1 . 10(-5) M. Losses of basal and anion-stimulated activities were similar at both ages. Fluoride and azide stimulations relative to basal activities were greater in particulates than in homogenates, while relative epinephrine activity was lower in particulates, suggesting qualitative alteration of adenylate cyclase during preparation of particulates. These studies show that adenylate cyclase activity in rat liver is presently best quantitated in homogenates and suggest caution in comparisons of enzyme activities based on particulates or membranes prepared from animals of differing physiologic states.


Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes. The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.


The calcium accumulation in a microsomal fraction from porcine coronary artery smooth muscle. A study of the heterogeneity of the fraction. 1. Microsomes prepared from the combined media and intima of pig coronary artery, take up Ca in an ATP-dependent way. This uptake is stimulated by oxalate. 2. Conditions have been determined to optimize the preparation of the microsomes in terms of their Ca accumulation activity. Careful homogenization of the tissue mince in 0.25 M sucrose by means of a Potter-Elvehjem homogenizer gives microsomal preparations with the highest specific activity for Ca accumulation. 3. Arguments are presented to support the hypothesis that, even in the absence of oxalate, Ca accumulation occurs into the lumen of the vesicles, and that these vesicles have a low Ca permeability. 4. Density gradient analysis shows that the microsomal fraction prepared from pig coronary artery media and intima is composed of vesicles that are heterogeneous in enzymatic composition. 5. Adenylate cyclase appears to be a predominantly plasma membrane-bound enzyme. Rotenone-insensitive NADH-cytochrome c reductase and choline phosphotransferase, two putative markers for internal membranes, give distinct banding patterns on on isopycnic centrifugation, indicating different intracellular localization. 6. There is a difference between the density gradient distribution pattern of Ca uptake measured in the presence or absence of oxalate. The latter coincides more closely with plasma membrane markers. The former resembles more the distribution of rotenone-insensitive NADH-cytochrome c reductase.


The nature of ABH blood group antigens in human gastric secretion. The origin of blood group ABH activity in human gastric content was investigated. Dialyzed and lyophilized samples of ten individual gastric secretions were assayed for ABH antigen under various conditions. The native activity persisted in delipidated residue of the respective secretions, but was completely missing in the lipid extracts of the analyzed samples. The alkaline degradation of the native and delipidated samples led to total loss of blood group activity of the analyzed materials, but no effect on A-active glycosphingolipid was evolved. Purified glycolipid portion of the lipid extract was lacking ABH activity and was shown to have distinct composition. This fraction contained only glyceroglucolipids and neither sphingosine nor other carbohydrates were present. On the basis of blood group activity assays of the native, delipidated, alkaline degraded samples and also on glycolipid analysis it was established that the ABH blood group activity of stomach secretion originated entirely from the glycoprotein portion of these samples.


Characterization of plasminogen activator in human cervical cells. Plasminogen activator activity was detected in human gynecologic specimens using a synthetic fluorogenic peptide substrate assay and confirmed by an 125I-labeled fibrin plate assay. Epithelial cells in these samples contain enzymatic activity that biochemically resembles both the well-characterized plasminogen activator, urokinase, and the less-specific plasminogen activator, trypsin. Inhibition of the cervical cell activity by diisopropylfluorophosphate and p-nitrophenyl-p'-guanidinobenzoate demonstrates that, like urokinase and trypsin, this plasminogen activator is also a serine protease. Polyacrylamide gel electrophoresis of plasminogen that had been incubated with cervical cells indicated the same mechanism of plasminogen activation as exhibited by urokinase. We attempted to correlate plasminogen activator activity of each sample with cytomorphologic diagnosis. Three of the four dysplastic samples analyzed showed higher plasminogen activator activity than did the normal samples.


Blood glycoprotein from antarctic fish. Possible conformational origin of antifreeze activity. High resolution 1H NMR and circular dichroism (CD) measurements have been performed on aqueous solutions of antarctic fish antifreeze glycoprotein. The carbohydrate contribution ot the observed CD spectrum has been estimated from closely analogous model compounds. The residual peptide contribution cannot be interpreted of the known spectral behaviour of alpha-helix, beta-sheet and random coil. Instead it resembles the CD spectrum of beta-structure in position, magnitude and spectral form, but is of opposite sign, indicating a specific but unusual peptide conformation, which we suggest may be stabilised by non-bonded interactions between the peptide backbone and the carbohydrate sidechains. Previous evidence which supports this interpretation is reviewed. NMR and CD measurements between -2 and +30 degrees C are consistent with conformational stability throughout the biologically relevant temperature range. The mechanism of the antifreeze activity is discussed in terms of the spatial and orientational correlations of sugar hydroxy groups and water in the liquid and solid states. The implication of an ordered peptide structure is explained by the comparison of the antifreeze glycoprotein with synthetic water-soluble polymers which also exhibit limited antifreeze properties.


[Biosynthesis of RNA in the nuclear structure of wilted cotton plant leaves]. The development of verticillate wilt in cotton plants is accompanied by an increase in the RNA-polymerase activity in isolated nuclear structures of cotton leaves at the latent stage of disease followed by its decrease when the plants develope inner signs of disease. The analysis of biosynthetic rates of nuclear RNA individual fractions showed that at the latent stage the biosyntheses of rRNAs and mRNAs in the nuclear structrues of the leaves are considerably enhanced. The development of inner signs of disease is accompanied by a disturbance of biosynthesis in the nucleus, primarily of mRNA. In the cells of wilted plants only 60% of the mRNA nucleotide sequences present in the controls are synthesized. Deficiency in matric cells for protein biosynthesis leads to further progress of the disease and to destruction of cells.


[Use of lipid exchange proteins for investigation of lipid dependence of rat liver cytochrome P-450]. The ability of lipid exchange proteins to introduce foreign phospholipids into intact microsomes was used for a study of the lipid dependence of rat liver cytochrome P-450. Cytochrome P-450 was converted into inactive cytochrome P-420 by treatment of rat liver microsomes with lysophosphatidylcholine. When such microsomes were incubated with phosphatidylcholine in the presence of lipid exchange proteins the lysophosphatidylcholine was partly replaced by phosphatidylcholine and the inactive cytochrome P-450 was reactivated. Similar incubation of the inactivated microsomes with phosphatidylethanolamine did not lead to reactivation of the enzyme.


[Effects of cycloheximide and chlorotetracycline on the activities of RNA-polymerases in mouse liver nuclei]. The activities of RNA-polymerases I and II in mouse liver nuclei isolated after injection of various poisons to mice were studied. An increased activity of RNA-polymerase I in the nuclei isolated from mouse liver after injection of the inhibitors of translation, i.e. cycloheximide and chlorotetracycline, was found. The activity of RNA-polymerase II remained unchanged. The inhibition of protein synthesis in mitochondria following the injection of chloramphenicol did not affect the activities of nuclear RNA-polymerases. The animal poisoning by carbon tetrachloride accompanied by a total decrease of the rates of protein and RNA synthesis in the liver decreased the activities of both RNA-polymerases, the activity of RNA-polymerase II being inhibited stronger as compared to that of RNA-polymerase I.


[Stabilization of modified glucose oxidase from Penicillium vitale incorporated into the polymeric chains of gels]. Glucose oxidase from Penicillium vitale was modified by unsaturated compounds, e.g. acrolein, allylisothiocyanate, acryloyl chloride and maleic anhydride. The degree of modification for the respective agents made up to 27.2, 8.3, 11.1 and 35.0% of the amine residues; the enzymatic activity was thereby retained by 98, 100, 0.03 and 58%, respectively. The thermal stability of modified enzymes was decreased 2-7 times. The modified preparations were copolymerized with acrylamide or 2-hydroxyethyl methacrylate, spatially cross-linked by N,N'-methylenebisacrylamide or ethyleneglycol dimethacrylate, respectively. The rate constants for thermal inactivation of immobilized glucose oxidase were 2-31 times lower as compared to the inactivation constants of the unmodified enzyme, incorporated into the same matrices.


[Energetics of active carbohydrate transport into mycoplasma cells]. Possible energy supply pathways of active transport of non-metabolized 3-O-methylglucose (3-O-mG) into Acholeplasma laidlawii cells are studied. 3-O-MG transport is sensitive to arsenate, inhibitors of glucolysis and membrane Mg-dependent ATPase (dicyclohexylcarbodiimide) and to uncoupling agents. It is suggested that glycolysis is the main energy source, which main enzymes are found in cell-free extracts. A. laidlawii cells are capable to glycolytic ATP synthesis using glucose as a substrate but not fructose, arginine and ethanol. ATP synthesis is also sensitive to arsenate and glycolytic inhibitors and is resistant to uncoupling agents, dicyclohexylcarbodiimide, valinomycin and nigericin. Probably, hydrolysis of glycolytic ATP by membrane-bound ATPase results in the formation on a membrane of both components of proton-motive force, which supply energy for the active 3-O-MG transport. It is supported with the data on the inhibitor analysis of transport using ionic-selecive antibiotics and lipid-soluble ions.