Acceptor-specific glucuronyl transfer catalyzed by beta-glucuronidase.

Highly purified rat liver microsomal or lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) catalyzes the specific transfer of glucuronly residues from phenyl-beta-D-[U-14C]glucuronide to acceptor sugars. Specificity requirements of acceptor sugars are found to be: pyranose structure, 4C1-conformation and equatorial position of C2 and C3 hydroxyl groups or pyranose structure, 1C4-conformation and equatorial position of C3 and C4 hydroxyl groups. The acceptor capacities of 30 monosaccharides and glycosides including di- and tri- saccharides conform to this prinicple. The specificity of the beta-glucuronidase catalyzed glucuronyl transfer is proved by the exclusive formation of beta-glucuronly (1--3)glycosidic linkages. Glucuronly transfer rates increase with increasing donor substrate and increasing acceptor sugar concentration. In the presence of 1 M acceptor sugar the ratio of the transfer rate to the rate of enzymatic hydrolysis is about 2:1. An 'acceptor substrate binding site' on the surface of the beta-glucuronidase molecule which brings the C3 hydroxyl function of the acceptor sugar close enough to the C1 atom of the glucuronyl residue, is postulated.


Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum.

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.


Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).


The digestion of the oxidized B chain of insulin by human neutrophile proteases: elastase and chymotrypsin-like protease.

The specificities of human neutrophile elastase and chymotrypsin-like protease towards oxidized insulin B chain were studied. The neutrophile elastase was found to differ from porcine pancreatic elastase in its specificity towards insulin B chain. The neutrophile elastase preferred mostly valine near the cleaved bond in contrast to pancreatic elastase which preferred alanine as well as valine near the cleaved bond. Human neutrophile chymotrypsin-like protease was found to cleave mostly bonds involving leucine and phenylalanine.


A new procedure to derive weighting factors for nonlinear regression analysis applied to enzyme kinetic data.

The experimental variance of enzymic steady-state kinetic experiments depends on velocity as approximated by a power function (Var(v) = K1 . valpha (Askelöf, P., Korsfeldt, M. and Mannervik, B. (1976) Eur. J. Biochem. 69, 61--67). The values of the constants (K1, alpha) can be estimated by making replicate measurements of velocity, and the inverse of the function can then be used as a weighting factor. In order to avoid measurement of a large number of replicates to establish the error structure of a kinetic data set, a different approach was tested. After a preliminary regression using a 'good model', which satisfies reasonable goodness-of-fit criteria, the residuals were taken to represent the experimental error. The neighbouring residuals were grouped together and the sum of their mean squared values was used as a measure of the variance in the neighbourhood of the corresponding measurements. The values of the constants obtained in this way agreed with those obtained by replicates.


Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.


Enzymic cis-trans isomerization of nitrofuran derivatives: isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes.

Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.


Insulin degradation. XXV. Glutathione-insulin transhydrogenase activity of rat liver and kidney during the development of streptozotocin-diabetes.

The activity of glutathione-insulin transhydrogenase (glutathione:protein-disulfide oxidoreductase, EC 1.8.4.2) in the liver and kidneys of rats during the development of streptozotocin-induced diabetes has been studied. Following a single injection of streptozotocin, the transhydrogenase activity fell rapidly for 7-8 days and then gradually with time in both organs. In contrast to the control rats where approximately 25% of the enzyme is in a 'latent' state, nearly all the transhydrogenase activity in the diabetic liver appears to be in the free or functional form. The results are consistent with the hypothesis that both hepatic and renal glutathione-insulin transhydrogenase activity are under feedback control by circulating insulin. The possibility is discussed that the latent state may represent a storage form of the enzyme, which in insulin-insufficiency states is mobilized to the free or functional form for cell function.


Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.


Esterification of exogenous and endogenous fatty acids by rat adipocytes in vitro.

Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.


Palmitoyl coenzyme A synthetase activation by uncomplexed ATP.

Uncomplexed ATP, ADP, alpha, beta-methylene ATP and adenosinetetraphosphate were shown to activate long chain fatty acyl-coenzyme A synthetase. The presence of uncomplexed nucleotides was shown: (1) to induce the palmitoyl-AMP-dependent palmitoyl-coenzyme A formation, (2) to activate the MgATP-dependent overall reaction and (3) to stabilize the synthetase against ageing denaturation. In all three cases the observed nucleotide activation was shown to be eliminated upon metal chelation.


Purification and properties of a lipolytic acyl-hydrolase from potato leaves.

A lipolytic acyl-hydrolase was purified 520-fold from an homogenate of potato leaves (Solanum tuberosum L. cv. Benimaru). The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.6 and a molecular weight of about 110,000. It had pH optima of 5.5 and 5.0, and Km values of 0.26 and 0.54 mM for monogalactosyldiacylglycerol and phosphatidylcholine, respectively. The pH dependences were altered by the addition of Triton X-100. No separation of these two hydrolyzing activities was achieved; the ratio of the specific activity of galactolipase to that of phospholipase (about 7/1) remained constant throughout the purification procedures. Both the activities were changed in parallel with each other by the addition of reagents and by heat treatment. The enzyme clearly catalyzed the deacylation of the several classes of galacto- and phospholipids. These results suggest that a single enzyme is responsible for both activities.


alpha-Hydroxylation of newly synthesised fatty acids by a soluble fraction from germinating pea.

A soluble fraction from germinating pea (Pisum sativum) seeds alpha-hydroxylated newly-synthesised fatty acids to form alpha-hydroxypalmitic and alpha-hydroxystearic acids. In contrast to fatty acid synthesis from [14C] malonyl CoA, alpha-hydroxylation was inhibited by exogenous phospholipids. alpha-Hydroxylation was optimal at pH 8, required reduced pyridine nucleotides and was inhibited by EDTA and imidazole.


Methyl-directed desaturation of arachidonic to eicosapentaenoic acid in the fungus, Saprolegnia parasitica.

The lipids of Saprolegnia parasitica contain 5,8,11,14,17-eicosapentaenoic acid as major constituent. No other acid having (n-3) structure was detected, but 5,8,11,14-eicosatetraenoic (arachidonic) acid and its common precursors of (n-6) structure are present in significant amounts. During rapid growth of the organism, [1-14C]acetate was efficiently incorporated into fatty acids. Arachidonic acid was labeled after 2 h to nearly the same extent as any precursor acid and 14C in eicosapentaenoic acid reached this level within 6 h. Results of incubations with labeled fatty acids indicated that, in S. parasitica, oleic, linoleic, (6,9,12)-linolenic and arachidonic acids are major intermediates in the pathway to eicosapentaenoic acid. Methyl-directed desaturation of (n-6) to (n-3) acids does not occur with C18 acids but is specific for the polyunsaturated C20 chain length. Arachidonic acid is the direct precursor of eicosapentaenoic acid.


Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.


A new cerebroside from Fusicoccum amygdali Del.

A homogeneous cerebroside was isolated from a strain of Fusicoccum amygdali Del., a fungus pathogenic to almond and peach. Chemical degradations, together with extensive application of nuclear magnetic resonance and mass spectrometry techniques, led to elucidation of its structure. This corresponds to N-2'-hydroxy-3'-trans-octadecenoyl-1-O-beta-D-glucosyl-9-methyl-cis-4,x-8-sphingadienine.


Phospholipase activities in subcellular fractions of human platelets.

A homogenate of human platelets was fractionated by zonal ultracentrifugation into membranes, various granules and mitochondria. The membrane fraction was composed of two populations. The first, which represented 75% of the proteins, was rich in plasma membranes; the second, which represented the remaining 25%, was rich in microsomal membranes. Lysophospholipase was essentially localised in the cytosol. Phospholipase A1 which was only weakly bound to membranes, was mostly found in the soluble fraction (75%); the remainder was located in the plasma membranes and the mitochondria. Two-thirds of the phospholipase A2 was found in the particulate fractions.


Short chain ceramides as substrates for glucocerebroside synthetase. Differences between liver and brain enzymes.

In order to increase the sensitivity of the assay for ceramide: UDPGlc glucosyltransferase, the enzyme that makes glucocerebroside, we synthesized a variety of ceramide homologues that might be better substrates than the naturally occurring ceramides. N-Octanoyl sphingosine proved to be the best lipid tested in liver and brain. It could be added to the tissue homogenate in the dry form, as a thin layer coated on Celite, or in liposomes, prepared from lecithin and cerebroside sulfate. The liposomal form produced better replication of assay values. It is suggested that the addition of cerebroside sulfate to liposomal preparations might be a good, and more physiological, replacement for the commonly used dicetyl phosphate. A new homologue of DL-sphinganine, decasphinganine, was synthesized by an efficient series of steps and acylated with different fatty acids to form ceramide homologues. The best substrate in this series was the lauroyl amide and it is suggested that this lipid be used in cerebroside synthetase assays because of the convenience of preparing it, even though it is not as good as octanoyl sphingosine. Both compounds are distinctly better than natural ceramide or DL-sphinganine amides. From comparisons of enzyme activity under various conditions, the tentative conclusion is drawn that the enzymes in liver and brain have different properties, and that liver has two different synthetases.


Glycerophospho-(N-acyl)-ethanolamine lipids in degenerating BHK cells.

The phospholipids, which accompany semilysobisphosphatidic acid from degenerating BHK cells, were identified as a mixture of glycerophospho-(N-acyl)-ethanolamine lipids. The identification was based on infrared spectroscopy, thin-layer chromatography and ethanolamine content of the intact lipids or their partial degradation products. Sequential treatments with mild acid and alkali revealed the presence of three different derivatives: the most abundant of these was the O-(1-alkenyl) ether derivative (plasmenyl-(N-acyl)-ethanolamine), which represented 55-60% of the total glycerophospho-(N-acyl)-ethanolamine lipids; the O-alkyl derivative (plasmanyl-(N-acyl)-ethanolamine) and the di-O-acyl derivative (phosphatidyl-(N-acyl)-ethanolamine) each represented about 20% of the total.


The mechanism of modification by propranolol of the metabolism of phosphatidyl-CMP (CDP-diacylglycerol) and other lipids in the rat pineal gland.

The mechanism underlying the alteration of phospholipid metabolism in rat pineal gland in vitro produced by propranolol and tertiary amine local anesthetics was investigated. 0.1 mM propranolol did not affect either the levels or specific activity of [32P]ATP in glands. In the presence of the drug, the incorporation of cytidine, but not of inorganic phosphate, into phosphatidyl-CMP (CDP-diacylglycerol) was dependent on the cytidine concentration. The incorporation of glycerol into phosphatidyl-CMP, phosphatidylinositol and phosphatidylglycerol was enhanced by propranolol, whereas labeling of phosphatidylcholine was decreased. When both 1 mM propranolol and 1 mM inositol were present, labeling of phosphatidylinositol was further increased, stimulation of phosphatidyl-CMP and phosphatidylglycerol labeling was reduced and incorporation into phosphatidylcholine and triacylglycerol was depressed. The incorporation of [3H]inositol into pineal lipids was also enhanced by propranolol. 10 microM propranolol inhibited rat liver phosphatidic acid phosphohydrolase by 50%, while local anesthetics were less potent in the decreasing order: dibucaine greater than tetracaine greater than lidocaine greater than procaine. The propranolol-induced accumulation of phosphatidyl-CMP was prevented by supplying adequate freely diffusible inositol in the medium. The phosphatidyl-CMP which accumulated was not utilized for the enhanced formation of phosphatidylinositol brought about by norepinephrine. The results indicate that propranolol and local anesthetics redirect pineal phospholipid metabolism in part by inhibition of phosphatidic acid phosphohydrolase.


Physicochemical and biological properties of glucagon-like polypeptides from porcine colon.

Polypeptide material displaying glucagon-like immunoreactivity was isolated from porcine colon using immunoaffinity chromatography. The immunoreactive material was tightly bound to high molecular weight proteins but was dissociated by 0.1% w/v sodium dodecyl sulphate solution into immunoreactive components of approximate molecular weights 12,000,8000,5000 and 3000. These components reacted at least 50 times more strongly with antibodies specific for the N-terminal region of glucagon than with antibodies specific for the C-terminal region of glucagon. While the 8000 and 3000 dalton fractions were homogeneous, the 12,000 and 5000 dalton fractions were resolved into multiple bands by isoelectric focusing. The 12,000 dalton fraction was devoid of glycogenolytic and lipolytic activity, was not insulin releasing and showed no ability to bind to receptor sites specific for glucagon on hepatic plasma membranes and to active hepatic adenylate cyclase. The 8000 and 5000 dalton components showed weak lipolytic activity. The possible significance of colonic glucagon-like immunoreactivity relative to pancreatic glucagon and immunoreactivity from other tissues is discussed.


Lactoperoxidase-catalyzed iodination of human IgM. Differences between 7 S IgM and 19 S IgM and between cell surface 7 S IgM and serum 7 S IgM.

We have studied the lactoperoxidase-catalyzed iodination of human IgM and have measured the ratio of radioactivity incorporated into the mu chain to that incorporated into the L chain (i.e. the mu/L ratio). Both 7 S and 19 S IgM were examined. The ratio of radioactivity was found to be larger for 7 S IgM than for 19 S IgM for all four of the monoclonal IgM proteins examined. The data suggest that some tyrosines of the mu chain which are buried and not available for iodination in 19 S IgM become exposed on conversion of 19 S IgM to 7 S IgM. The mu/L ratio for the IgM found on the cell surface of RPMI 8392 cells was significantly smaller than the ratios for all of the five 7 S IgM proteins studied in solution. It appears, therefore, that a portion of the mu chain of the cell surface IgM of the RPMI 8392 cells is buried in the membrane.


Separation of cathepsin B1 and related enzymes from rat skeletal muscle.

Rat muscle was extracted at pH 4 and submitted to gel-filtration on Sephadex G-75 and to chromatography on DEAE-Sephadex. Gel-filtration gave a large peak of activity towards Bz-Arg-NNap with an estimated molecular weight of 25,500. Activity towards Bz-Arg-NH2 was present in this peak and in another peak of molecular weight 45,000. The second peak also hydrolysed benzoyl-glycyl-L-arginine. DEAE-Sephadex gave five peaks of Bz-Arg-NNap hydrolysing activity; all showed thiol dependence. Peaks III, IV and V hydrolysed Z-Ala-Arg-Arg-NNap-OMe rapidly; they also inactivated aldolase and were strongly inhibited by leupeptin. They are probably isoenzymes of cathepsin B1. Peak I showed these properties to a relatively small extent. 7-(N-Benzoyl-DL-argininamide)-4-methylcoumarin appears to be an alternative substrate for cathepsin B1; it was hydrolysed also by peak I, but relatively less rapidly. Peaks I and II were inhibited more than peaks III, IV and V by a muscle extract. Total activity of the Bz-Arg-NH2-hydrolysing enzyme in extensor digitorum longus muscle increased after denervation.


Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.


Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.


Circular dichroism and conformational transitions of leucoagglutinin.

Conformational transitions of leucoagglutinin from kidney bean (Phaseolus vulgaris) were studied by the circular dichroism (CD) probe. The CD spectrum of leucoagglutinin in the far ultraviolet zone was similar to the spectrum of concanavalin A, indicating the presence of high amount of the beta pleated sheet structure. The CD spectrum of the leucoagglutinin in the near ultraviolet zone was similar to the CD spectra of peanut and soybean agglutinins. The conformation of leucoagglutinin was not significantly affected by alkali at pH 9.4 or 10.4; however, at pH 11.2 the conformation was disrupted. Appearance of a new CD band at 255 nm was related to ionized OH groups of tyrosine side chains. Acidification of the strongly alkaline solutions leads to a loss of all near ultraviolet bands and formation of large amounts of pleated sheet structure in this denatured lectin. Native leucoagglutinin was resistant to acid, as only a slight weakening of the CD bands was observed at pH values as low as 2.1 to 2.4. These changes (at pH 2.5--2.8) were facilitated by EDTA, and the demetallized lectin slowly precipitated from these solutions. Sodium dodecyl sulfate (SDS) disorganized the native structure of leucoagglutinin, and the secondary structure was reorganized into new conformations of moderate alpha-helix contents. These transitions were strongly promoted by acid, enhancing the helix content to about 30%.


The reactivity of tryptophan residues in proteins. Stopped-flow kinetics of fluorescence quenching.

The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues.


A highly reactive tyrosine residue as part of the indole and benzodiazepine binding site of human serum albumin.

The interaction of L-tryptophan and four benzodiazepine derivatives with tyrosine-modified human serum albumin was investigated by equilibrium dialysis and circular dichroism measurements. Out of the 18 tyrosine residues of the human serum albumin molecule, only 9 could be modified with tetranitromethane. At least up to a degree of modification of 5, the conformation of human serum albumin was not changed and no crosslinking and fractionation has been found, as revealed from circular dichroism measurements in the far ultraviolet range and from SDS polyacrylamide electrophoresis. The modification of only 2 out of the 9 accessible tyrosine residues of human serum albumin strongly affects the binding of L-tryptophan and diazepam to their common, stereospecific bindining site. This was evidently shown by a reduction of the association constants by more than 90% and by a large reduction of the extrinsic Cotton effects of four benzodiazepines bound to human serum albumin. The numbers of binding sites remained unchanged. Strong evidence was presented that only one tyrosine residue, which reacts faster with tetranitromethane than all others, is mainly involved in the specific indole and benzodiazepine binding site of human serum albumin. The location of this highly reactive tyrosine residue and that of the specific indole and benzodiazepine binding site within the human serum albumin primary structure is discussed.


Specific fragmentation of human erythrocyte spectrin by chemical cleavage at cysteine residues.

Spectrin, isolated from human erythrocyte membrane, was specifically cleaved at the amino side of its cysteine residues by reacting it with 2-nitro-5-thiocyanobenzoic acid at pH 8.0 and incubating the product at pH 9.0. Conditions were developed to obtain quantitative cleavage, with virtually no side reactions due to exposure to the alkaline pH. The solubility and aggregation state of the spectrin fragments in 0.2 M sodium chloride, in 7 M guanidine hydrochloride or in 10 M urea, at pH 8.0, allow separation and partial purification of the fragments by gel filtration or by ion-exchange chromatography. Our results strongly suggest that various parts of the spectrin molecules have similar amino acid compositions. Due to the relatively limited number of fragments, this cleavage method is a promising tool for further elucidation of the structure of spectrin and for understanding its role in the erythrocyte membrane.


Galactose oxidase. An enzyme with lectin properties.

Galactose oxidase interacts with immobilized D-galactosyl residues and related immobilized and free sugars under the conditions of affinity electrophoresis in polyacrylamide gel and agglutinates sialidase-treated human erythrocytes. The agglutination is also inhibited by D-galactose and its derivatives and is temperature dependent. The sugar binding and hemagglutinating activity are preserved after removal of Cu2+ essential for enzymic activity. These properties are very similar to those of some typical lectins; however, a number of D-galactose specific lectins do not possess any detectable galactose oxidase activity.


Properties of Ulex europaeus II lectin isolated by affinity chromatography.

A lectin was isolated from Ulex europaeus seeds by affinity chromatography on affinity adsorbent prepared by copolymerization of acrylamide, N,N'-methylene bisacrylamide and maleylated hog stomach peptone. The lectin is homogeneous as judged by ultracentrifugation (s20,w = 6.4 S), electrophoretic and gel chromatography criteria; it contains 4.2% neutral sugar and 1.4% glucosamine. Its molecular weight is approx. 110,000 and the molecule consists of two noncovalently linked protomers which are formed by two covalently bound basic subunits (Mr = 30,000). The preparation contains three isolectins differing in the strength of interaction with specific sugars (cellobiose, N-acetyl-D-glucosamine) under the conditions of affinity electrophoresis. The lectin is non-specific with human ABO blood group system, the agglutination is inhibited by partial chitin hydrolysate, hog stomach peptone and high concentration of cellobiose.


Extensive carboxypeptidase digestion of clam alpha-paramyosin. The effect on the size and solubility of the residual protein.

The purpose of this paper is to provide further evidence that the C-terminal 1/3 of the alpha-paramyosin molecule is the portion responsible for the low solubility of alpha-paramyosin at neutral pH and low ionic strength. This was accomplished by digesting from the C-terminal end with carboxypeptidases A and B in 2 M urea at pH 8.5. The solubility increased as the molecular weight decreased until a stable segment 2/3 of the size of the molecule remained.


Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients.


Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.


Complete amino acid sequence of the myoglobin from the Pacific sei whale, Balaenoptera borealis.

The complete amino acid sequence of the major component myoglobin from Pacific sei whale, Balaenoptera borealis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apomyoglobin, over 75% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by the sequence analysis of peptides that resulted from further digestion of the amino-terminal and central cyanogen bromide fragments. The amino-terminal fragment was specifically cleaved at its two tryptophanyl residues with N-chlorosuccinimide and the central cyanogen bromide fragment was cleaved at its glutamyl residues with staphylococcal protease and at its single tyrosyl residue with N-bromosuccinimide. The primary structure of this myoglobin proved identical with that from the gray whale but differs from that of the finback whale at four positions, from that of the minke whale at three positions and from the myoglobin of the humpback whale at one position. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.


Purification and characterization of a major component from the cytoplasmic matrix of cultured murine L cells.

This study describes the purification and preliminary characterization of a 94 000 molecular weight protein (S-94) previously known only as a major band in SDS-polyacrylamide gels of total proteins from cultured murine cells. Native S-94 is a soluble constituent of the cytoplasm and sediments at 5.0 S in sucrose gradients, behavior compatible with that of a 94 000 molecular weight monomer. S-94 is purified more than 20-fold from the 100 000 X g supernatant of murine L or L1210 lymphoma cells by DEAE-Sephadex (A-50) chromatography in the presence of 2-mercaptoethanol followed by Sephadex G-200 chromatography in 8 M urea. The protein prepared by this procedure is extensively aggregated, but is essentially homogeneous by SDS-polyacrylamide gel electrophoresis. S-94 preparations from the two types of murine cells behave identically during the purification procedure and are presumed to be the same protein. It appears that these cells contain only one major protein of 94 000 molecular weight. Purified S-94 yields a distinctive pattern of fragments following CNBr degradation, including one peptide of 36 100 molecular weight. The amino acid composition is distinguished by being relatively rich in threonine, glycine and lysine, but poor in valine, leucine and tyrosine.


Laser Raman spectroscopy of acid phosphatase from rat liver after neuraminidase treatment.

The conventional Raman technique was used to obtain spectra of acid phosphatase isoenzymes I and II from rat liver after neuraminidase enzyme treatment for 24 h. The low frequency bands below 300 cm-1 which were observed in native lyophilized isoenzymes are obtained from the sialic acid groups of these glycoproteins. These bands disappeared after neuraminidase treatment and the broad bands at 310, 260, 200 and 80 cm-1 are obtained from protein modes. The small differences between the Raman spectra of these isoenzymes disappeared after the neuraminidase treatment.


Lanthanide ion probes of structure in biology. Environmentally sensitive fine structure in laser-induced terbium(III) luminescence.

The 488 nm line of the CW argon ion laser provides a convenient visible source for the direct excitation of the emissive 5D4 state of the Tb(III) ion. Room temperature emission spectra of Tb(III) in a variety of environments have been examined under relatively high resolution. The samples studied include structurally well-characterized crystalline solids, model chelate complexes in solution and Tb(III) bound to the enzyme thermolysin and the protein parvalbumin. The fine structure in the emissions is caused by ligand field splittings of both ground and excited state J manifolds. These spectra provide signatures sensitive to the immediate coordination environment of the Tb(III) ion. Solid state/solution state structural comparisons are made. The emission fine structure reveal differences between the EF side calcium-binding sites of parvalbumin and the calcium site 1 of thermolysin.


Studies on factor VIII-related protein. II. Estimation of molecular size differences between factor VIII oligomers.

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.


Plant histone 2 from wheat germ, a family of histone H2a variants. Partial amino acid sequences.

1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.


The N-terminal sequence of the ribosomal 'A' protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 11227).

The 'A' protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The 'A' protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the 'A' proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the 'A' protein from both moderate halophiles showed substantial homology to EL 12 (75--80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli were also isolated and their amino acid compositions determined.


Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.


Isolation and partial characterization of human factor V.

Factor V was isolated from human citrate plasma by very mild purification steps. Cryoprecipitation, fractionation with polyethylene glycol 6000, gel filtration of AcA 44 and adsorption of haptoglobin to immobilized hemoglobin were applied successively, resulting in factor V preparations with a specific activity of 14.5 unit/mg. The yield was 28 percent. A molecular weight of 296 000 was determined by gel filtration and the apparent sedimentation constant found by ultracentrifugation in a sucrose gradient was 7.8 S. Parallel experiments with citrate plasma resulted in the same molecular weight and sedimentation constant. Polyacrylamide gel electrophoresis of factor V in the presence or absence of sodium dodecyl sulfate showed a single protein band. Incubation with human thrombin resulted in an 8-fold activation of the purified factor V.


The role of crotoxin subunits in tropical rattlesnake neurotoxic action.

The major toxin (crotoxin) of Crotalus durissus terrificus (neotropical rattlesnake) is known to be a reversible non-covalently associated complex consisting of an acidic and basic subunit. On separation biological activity is found only with the basic subunit, yet, although void of detectable biological activity, the acidic subunit is essential for the full neurotoxic activity of the complex. Recent evidence suggests that crotoxin A serves as a 'chaperone' to enhance the specificity of crotoxin B and, upon binding, crotoxin A is released to the medium. This study was designed to test this hypothesis. Dimethyl suberimidate, a bifunctional cross-linking agent, was used to irreversibly bind the two subunits. Disc electrophoresis, ion-exchange chromatography, molecular sieve chromatography, capillary isotachophoresis and isoelectric precipitation confirm the existence of an inter-subunit covalently cross-linked complex. The conversion of a dissociable complex to a non-dissociable complex abolished neurotoxicity. Although neurotoxicity was lost, phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4), which is found associated with many presynaptic neurotoxins, was unaffected. The data in this paper add credence to the 'chaperone' concept of crotoxin A and the importance of the reversible nature of the complex for full expression of neurotoxicity.


Preparation and properties of polyethylene glycol-trypsin adducts.

The covalent attachment of polyethylene glycol of 5000 daltons to non-essential groups on trypsin produces an adduct that no longer precipitates with anti-trypsin antibody. In comparison with trypsin, polyethylene glycol-trypsin preparations show equal or greater activity against N-alpha-benzoyl-L-arginine ethyl ester, about one-fourth activity against angiotensin II, and little activity against bovine liver catalase. The polyethylene glycol-trypsin adduct dissolves soft blood clots at one-fourth the rate of trypsin. Soybean trypsin inhibitor produces two-thirds inhibition of the adduct under conditions that cause complete inhibition of trypsin.


Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.


Two-dimensional gel electrophoresis of 125I-labeled surface proteins of human fibroblasts.

Human fibroblasts were labeled by the 125I-lactoperoxidase technique and the sodium dodecyl sulfate soluble proteins examined by two-dimensional gel electrophoresis to determine the charge heterogeneity of the surface proteins and test for differences in surface proteins in several hereditary disorders. Approximately 80 polypeptides were observed. Those below 65 000 daltons tended to occur as single spots, while those of higher molecular weight were often present as a series of polypeptides of similar molecular weight (charge isomers). The possible role of these proteins in cell-cell recognition is discussed.


Sodium dodecyl sulfate-protein polypeptide complexes in 8 M urea with special reference to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The effects of 8 M urea on the complexes formed between sodium dodecyl sulfate and protein polypeptide were found to be as follows: (1) The maximum amount of SDS bound is reduced by almost half, and the minimum equilibrium concentration of SDS necessary to reach saturation was nearly doubled; (2) The apparent content of alpha-helical structure deduced from CD measurement is reduced to only 50--70% of that in the presence of sodium dodecyl sulfate alone; (3) The effective size of the sodium dodecyl sulfate-protein polypeptide complex deduced from viscosity measurements is increased, but is still smaller than the effective size of the protein in 8 M urea alone.


Conformation change in reconstituted thin filament studied with fluorescence energy transfer between epsilon-ADP bound to F-actin and NBD-Cl bound to troponin-C.

The interaction of troponin and F-actin was studied by the methods of fluorescence energy transfer. We used epsilon-ADP bound to F-actin as a fluorescence donor and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to troponin-C in troponin as a fluorescence acceptor. The efficiency of the fluorescence energy transfer of this system was larger in the absence of calcium ion than in the presence of calcium ion. This shows that the interaction of troponin with F-actin changes in response to the concentration of calcium ion.


Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum.

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg2+ or Mn2+, but is inhibited by Ca2+. The Km is 0.5 mM ATP, similar to the Km of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a beta 2-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 . 10(-11) M, while glucagon was effective only at 1 . 10(-6) M. Prostaglandin E1 and E2 have a biphasic effect with activation of adenylate cyclase at 1 . 10(-5) M and a small but significant inhibition of enzyme activity at 1 . 10(-11) M.


Distribution of non-electrolytes in Halobacterium cells. I. Halobacterium marismortui.

Halobacterium marismortui is an obligatorily halophilic species isolated from the Dead Sea. When inulin, fructose or glycerol are added to suspensions of bacteria, the amounts of these substances recovered from centrifuges pellets are more than could have been present in the extracellular space. Thus a certain amount becomes associated with the bacteria, though not enough to equilibrate with all the cell water. The inulin or fructose concentration found after uptake of these substances was correlated with the cell sodium concentration. It is argued that inulin, fructose or glycerol is unlikely to be adsorbed on the outside of the bacteria and more probably crosses the plasma membrane. A possible scheme for explaining the data is presented.


Synthesis of protein, nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions.

The mixing of cyanamide and KNO2 produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O2. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO2 reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160,000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.


Stimulation of renal gluconeogenesis by angiotensin II.

Renal gluconeogenesis was studied in suspended tubule fragments isolated by collagenase treatment of rat kidney cortices. Angiotensin II increased glucose formation from pyruvate, lactate, and to a lesser extent from oxoglutarate and glutamine, but not from other substrates such as malate, succinate, dihydroxyacetone or fructose. Stimulation was significant with peptide concentration exceeding 1 . 10(-8) M and was also shown with an 8-Sar derivative. Other peptides such as 4-Ala-8-Ile-angiotensin II, hexapeptide and bradykinin had no effect. The stimulatory action of angiotensin II was additive to that of L-lysine, and 3',5'-adenosine cyclic monophosphate, suggesting a different mechanism of action. In the presence of maximally stimulatory concentrations of oleate, phenylephrine and 3',5'-guanosine cyclic monophosphate, however, the stimulatory effect of angiotensin II was absent. Cyclic GMP levels, however, did not increase in tubules after angiotensin II and phenylephrine addition, making a messenger function of this nucleotide unlikely. Omission of Ca2+ from the medium markedly reduced basal gluconeogenesis but did not result in a complete loss of angiotensin II effect. Reduction of medium potassium to 2 mM, however, increased basal gluconeogenesis and blunted the peptide effect. 1 mM ouabain was also able to inhibit the stimulatory effect of angiotensin II. Therefore changes in intracellular potassium levels are discussed as a possible mechanism of angiontensin action, whereas calcium seems not to be specifically linked to this metabolic action of angiotensin on the proximal tubule.


Kinetics of the glucocorticoid hormone-receptor interaction. False association constants determined in slowly equilibrating systems.

The usual way of in vitro determination of association constants for hormone-receptor complexes is criticized. It is shown that if incubation time is short, relative to the half-life of the hormone-receptor complex, the value of the apparent Ka is proportional to the time of incubation. No sign of lack of equilibrium is apparent from the Scatchard plots. The case of rapidly denaturing receptor molecule is also discussed, with similar conclusions. Although terminology and examples are taken from the field of the glucocorticoid receptor research, all deductions are valid for other systems with similar association (and denaturation or monomolecular transformation) mechanisms and kinetic parameters.


Microinjection of ricin entrapped in unilamellar liposomes into a ricin-resistant mutant of baby hamster kidney cells.

Fluorescein-labelled Ricin was entrapped in unilamellar liposomes; 14 microgram of protein was entrapped by 1 mg of lipids. Liposomes added to cells in culture in low serum medium can deliver entrapped Ricin to a Ricin-resistant mutant of baby hamster kidney(BHK)cells. Ricin entrapped in unilamellar liposomes inhibits protein biosynthesis at a concentration of 1.75 microgram/ml in Ricin-resistant cells. Ricin dissolved in medium at 50 microgram/ml does not affect protein synthesis in these cells.


The occurrence of cystinylglycine in blood plasma.

Cystinylglycine has been identified as a minor ninhydrin-positive component of deproteinized blood plasma from human, bovine, rat and rabbit blood. The amount present in human blood plasma is approximately one-fifth of that of cystine and is not significantly correlated with age or with the cystine level.


Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.


Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.


[Myoplasmic fixed charges of the barnacle muscle fiber (author's transl)].

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and phi X, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061--1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced phi X, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100 g KCl concentration in the external medium equal to 0.15 M, phi X was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in phi X of 20--50% depending on E1KCl] in the external solutions. The addition of ATP, Mg2+, and Ca2+ also causes a reduction of phi B by 30--50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when CKl is replaced by Nal. It also suggests a reduction of phi X during muscular contraction.


Interaction of metallochromic indicators with calcium sequestering organelles.

Examples are presented of the interaction between cell organelles and metallochromic indicators used in the measurement of ionized Ca2+. Sarcoplasmic reticulum was found to sequester murexide type indicators along with Ca2+ in the presence of ATP, but not to sequester arsenazo III and antipyrylazo III. The presence of a permeable anion suppresses the sequestration of murexide type indicators by the sarcoplasmic reticulum. In the presence of ruthenium red, both rat liver and beef heart mitochondria release sequestered Ca2+ with arsenazo III, but not with murexide.


Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.


Pattern of RNA synthesis during the regeneration by Owenia fusiformis (polychaete annelid).

The binding of [3H] Uridine labelled RNAs and [32P] RNAs to poly U filters using the Sheldon et al. technique (1972) shows an increase in the poly A+ RNA populations from total RNAs and cytoplasmic RNAs in the prereplicative phase of the regenerating process in Owenia fusiformis. This increase confirms that the first stage of this process consists in an activation of the transcription of the genetic material concerning mainly mRNAs synthesis activation.


[Cytidine and deoxycytidine aminohydrolase activities from Zea mays L. aerial parts: probable existence of two isozymes both of which possess the two activities].

Enzymatic proteins with deoxycytidine and cytidine aminohydrolase activities were partially purified from Zea mays L. aerial parts by using ammonium sulfate fractionation, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose.


[Characterization of histones and chromatin of the hepatopancreas in Palaemon serratus (Crustacea Natantia)].

The chromatin of shrimp hepatopancreas has been extracted from isolated nuclei and characterized. Nuclei were prepared in the presence of Cu++ and phenyl methyl sulfonyl fluoride in order to inhibit the nuclease and protease activities throughout the different purification steps. The purified nuclei are heterogenous in size and show a density of 1,367 g/ml determined on saccharose - glucose gradients. After washing in 0,14 M NaCl and then in 10(-2) M Tris-HCL, pH = 7,6, the nuclei were disrupted in water. The solubilized chromatin was precipitated in 0,15 M.NaCl. This chromatin is characterized by a high level of RNA (RNA/DNA = 0,38) and of non histone proteins (NHP/DNA = 0,6). The denaturation curve showed only one Tm at 69 degrees in 2.10(-4) M.EDTA. When the chromatin was extracted in the presence of staphylococcal nuclease, the Tm reached 80 degrees C. The kinetics of the digestion by the staphylococcal nuclease have been studied and show that 10 per cent of hydrolysis occurs within the first minute. The repeat length of DNA as determined with the polymers of higher order is 189 +/- 5 base pairs. The existence of nucleosomes was confirmed by electron microscopy. The superstructure of chromatin was not completely destroyed after solubilisation with a Potter. The histones were studied by gel electrophoresis after differential staining. The most important feature consists in the presence of two H1, two H2A and two H4. The acetylation levels of the histones were followed after injection of 14C-acetate in vivo. The subfraction H1, 0 was acetylated. Only one H3 was present and the two H2A fractions showed the same level of acetylation. H2B migrated faster than the H2A fractions like in Echinoderms. The two H4 fractions corresponded to two differently acetylated forms. Shrimp hepatopancreas histones were fractionated by molecular sieving on Biogel P 100 and characterized according to their electrophoretic properties as well as their amino-acid content. The amino-acid compositions of the different histone fractions were nearer to Echinoderm and Sipunculid histones, than Calf thymus homologue histones. All the fractions show a weaker basicity. The H3 fraction was the only one showing a lesser variability when compared to Calf thymus H3. The non histone proteins were extracted in 10(-2) M Tris-HCL, pH = 8 and 0.1 per cent SDS. A series of 50 proteins was detected. 80 per cent of the total amount of protein was localized in a molecular weight range comprised between 40 000 and 80 000 daltons. These proteins were compared to the histones and total proteins of sonicated chromatin solubilized by SDS in order to detect proteasic effects.


Prostaglandins E2 and F alpha levels in white and brown adipose tissues of cold acclimated rats as measured by a new micromethod.

A new micro-method was used to determine the effects of cold acclimation of rats on the levels of prostaglandin E2 and F alpha in both white and brown adipose tissues. Whereas PGF alpha levels were significantly higher than PGE2 levels in white fat, no difference between the amounts of the two prostaglandins was observed in brown fat. In both tissues, cold acclimation did not induce any change in prostaglandin levels.


[Phospholipase A2 activity in rat platelets].

Rat blood platelets show phospholipase A2 activities twenty times higher than human platelets. The breakdown of PE at pH 7.2 is linear for 15 minutes. There is no degradation at pH less than 5; the maximal activity is in the pH range 5.5-7.5. The presence of Ca2+ increases the phospholipase activity; an excess is not inhibitory. The optimal activity is obtained with 16 microM substrate concentration. Substrate inhibition is observed when the concentration exceeds 25 microM.


Use of p-aminophenyl D and L-lactic acids and p-aminophenyl pyruvic acid as effectors in the affinity chromatography of lactate dehydrogenase.

p-Aminophenyl pyruvic acid and D-p-amino-phenyllactic acid were immobilized on a new synthetic acrylic carrier bearing acylating N-succinimidyl ester groups. The derivatives obtained were used successfully to purify lactate dehydrogenase (LDH) by affinity chromatography, the elution being carried out by means of NADH or preferably L-phenyllactic acid. Moreover, the specific activity of the LDH contained in a human blood serum was increased 270 times, using L-p-aminophenyllactic acid immobilized on a mixed polyacrylic agarose carrier.


Protein synthesis in liver, muscle and gill of mullet (Mugil cephalus L.) in vivo.

1. Protein synthesis in mullet was measured by use of pulse injections of 14Cleucine into the hepatic portal circulation and by 4-hr continuous infusion of 14Ctyrosine into the dorsal aorta. 2. Liver protein synthesis was 37 mg/day per g wet weight of tissue. Plasma proteins accounted for about 25% of total liver protein synthesis. Replacement rate for intrahepatic proteins was 20%/day. Average polypeptide chain synthesis time was 3 min. 3. Gill protein synthesis was comparable to that in liver with a replacement rate of 23%/day. 4. Muscle protein synthetic rate was 0.54%/day, comparable to rates in rabbit and sheep when temperature difference is taken into account. 5. Constant infusion is shown to be a feasible technique for measurement of protein metabolism of fish in vivo.


The cytochemistry of Limulus eggs.

Cytochemical studies on uninseminated mature eggs of Limulus demonstrate the presence of carbohydrates, lipids and proteins in the egg envelopes and yolk. The vitelline envelope, cortical region and yolk are rich in 1,2-glycols, with the vitelline envelope, containing fewer reactive 1,2-glycol groups than other components of the egg. Neutral mucopolysaccharides are found in the cortical region and yolk, but only the cortical region of the eggs demonstrate the presence of sulfated mucosubstances (which are in part glycoprotein in nature) and glucose-6-phosphatase. Protein is evident in all egg components. Biochemical analysis demonstrate the protein in the egg envelopes of uninseminated eggs is composed of sixteen amino acids while that of developing eggs contain seventeen amino acid residues. Electrovalent linkages and non-S-S- covalent linkages between protein chains are shown to be instrumental in maintaining the stuctural integrity of Limulus egg envelopes. Neutral lipids, unsaturated lipids, phospholipids and fatty acids are demonstrated in yolk bodies and lipoproteins, unsaturated lipids and fatty acids constitute part of the egg envelopes. DNA is concentrated in the cortical region and the yolk bodies


Role of aggregation factor and cell type in sponge cell adhesion.

A pair of sponge species, Microciona prolifera and Halichondria bowerbanki, which lack mutual species specificity in their aggregation "factor", are useful in establishing the mechanisms of action of these factors. These sponges were dissociated both mechanically, which leaves the factor on the cell surface, and by Humphrey's (1963) method, which isolates the factor from the cells. The adhesive specificities which arose, in the various combinations tested, point to an intercellular factor bridge consisting of a single symmetrical unit. An analysis of most other workers' results is consistent with this interpretation. However, MacLennan and Dodd's (1967) results using other species would require a bridge consisting of two or more asymmetrical units. Differences were found in the specificity of adhesion of various types of cells within a single species. This presents a heretofore unconsidered problem in assesing the adhesive factor's mechanism of action. Three structurally distinct cell types were separated from a suspension of dissociated Microciona cells by velocity sedimentation. These cells differ greatly in adhesiveness. The differences in adhesion are correlated with numbers and positions of cells incorporated into aggregates. Such differences are considered in explaining the mechanism of action of the factors.


Toxicity and pharmacology of extracts from dorid nudibranches.

1. Aqueous extracts of digestive glands of specimens of the dorid nudibranchs Cadlina flavomaculata, Doriopsilla albopunctata, Anisodoris nobilis, Archidoris montereyenis, and A. odhneri were lethal when injected into shore crabs and when injected intraperitoneally into mice. 2. Aqueous extracts of the degestive glands of Doriopsilla albopunctata and of Anisodoris nobilis were shown by bioassay (guinea pig ileum)and by chemical determination to contain histamine. The amount present was far too small to account for the toxicity of the glands. 3. Extracts of the digestive glands of Anisodoris nobilis were fractionated by column chromatography on Biogel P-2 to yield an active fraction designated "dorid toxin". This produces lethargy and bradycardia in mice. In anesthetized rats it produces sustained (60 min or more) bradycardia and hypotension. On isolated hearts, especially spontaneously beating guinea pig atria, it has negative inotropic and chronotropic effects. 4. Dorid toxin has a molecular weight under 8000. It is heat stable and is not destroyed by trypsin, chymotrypsin or Pronase. It is therefore unlikely that it is a polypeptide.


Antennular sensilla of the brine shrimp, Artemia salina.

1. Scanning electron microscopy was used to characterize the external morphology of setae found on the antennules of adults and nauplii of the brine shrimp, Artemia salina (L.). The permeability of the antennular setae was studied by means of Slifer's crystal violet method. 2. Each antennule of an adult brine shrimp possessed a terminal cluster of sensory setae. Within a cluster there were two morphologically distinct kinds of sensilla, here designated type 1 and type 2. Three type 1 sensilla were observed on every antennule examined. The number of type 2 sensilla per antennule was usually four or five. 3. Type 1 sensilla of adults were 43 to 80 micrometer long and simple in external morphology. They were widest at the base, decreased in diameter gradually, and terminated as a finely tapered tip. No pores were resolved by scanning electron microscopy. 4. Type 2 sensilla of adults were shorter (shaft length, 12 to 23 micrometer) and displayed a single pore at the tip (average pore diameter, 0.4 micrometer). In thin section they were seen to possess a distinctive articular specialization of the cuticle at the base of the seta. 5. Dye penetration experiments indicated that type 2 sensilla were permeable to aqueous crystal violet, whereas type 1 sensilla were not. 6. The antennular setae of nauplii resembled type 1 sensilla in general shape, in being impermeable to crystal violet, and in lacking a terminal pore and basal articular specialization. Moreover, a total of three setae was normally present on each naupliar antennule, and the same number of type 1 sensilla was found on each adult antennule examined. If the three naupliar setae represent a developmental stage in the formation of three adult sensilla, available observations suggest that the larval setae are developmentally related to type 1, rather than to type 2 adult sensilla.


Development of vestigial tail muscle acetylcholinesterase in embryos of an anural ascidian species.

1. The ascidian Molgula arenata produces an anural larva lacking a tail and other structural features of typical urodele larvae in the family Molglidae, yet its embryos developed a histochemically detectable acetylcholinesterase in the tail muscle rudiment. Development of the myoblasts seemed to fail during the neurula stage. 2. Larval enzyme activity occurred at a mean of 5--6% of the level found in the urodele species Molgula occidentalis and Molgula manhattensis, as measured by scanning integrating microdensitometry of the histochemical reaction product. Some anural larvae had as much as 20% of the enzyme activity in urodele larvae. 3. This example of vestigial expression in the absence of other urodele larval features further illustrates the autonomy of a histospecific enzyme development thought to be controlled by an egg cytoplasmic determinant. Partial suppression of the determinant might be the cause of this diminished expression. 4. Two other anural molgulid species, Molgula occulta and Bostrichobranchus pilularis, did not have vestigial larval enzyme and possibly have lost the determinant completely.


The internal representation of solid shape with respect to vision.

It is argued that the internal model of any object must take the form of a function, such that for any intended action the resulting reafference is predictable. This function can be derived explicitly for the case of visual perception of rigid bodies by ambulant observers. The function depends on physical causation, not physiology; consequently, one can make a priori statements about possible internal models. A posteriori it seems likely that the orientation sensitive units described by Hubel and Wiesel constitute a physiological substrate subserving the extraction of the invariants of this function. The function is used to define a measure for the visual complexity of solid shape. Relations with Gestalt theories of perception are discussed.


Initial processing of visual information within the retina and the LGN.

The initial stage of information processing by the visual system reduces the information contained in the continuous image on the retina into a discrete set of responses which are carried from the lateral geniculate nucleus (LGN) to the visual cortex. -- 1. The optimal sampling of the light intensity distribution in the visual environment is achieved only if each channel in the visual pathways carries undistorted information corresponding to an image element. The visual system approaches as closely as possible the scheme of optimal spatial sampling, retaining the full information on the low spatial frequency content of the object light intensity. The ideal receptive field of a sustained LGN cell is then of the form J1(Kr)/Kr. -- 2. The experimentally determined receptive fields of sustained LGN cells (and to some extent retinal ganglion cells as well) in cat closely resemble the functional form J1(Kr)/Kr. The centre-surround organization of the receptive fields is therefore understood as a scheme which leads to a maximal information flow through the visual pathways. -- 3. The optimal sampling scheme cannot be realized by the retina alone, because of restrictions on the size of neural networks. It is therefore constructed in two stages, ending at the LGN level. A recombination of ganglion cell signals into optimal receptive fields is a major role of the LGN.


Generation of Gaussian noise with improved quasi-white properties.

Problems of hitherto used and suggested random signals for the identification of non-linear systems are reviewed. Two processes for the practical generation of random signals are proposed which avoid most of the shortcomings of the former test signals.


Human eye tracking as a sequential input adaptive process.

Sequential input adaptive system theory is applied to human eye tracking of targets with one-dimensional motion defined by since functions, sums of sine functions and triangle functions. The sequential theory describes the mean eye tracking movements of two subjects for these inputs. The theory is predictive for irregular inputs in that when its parameters for a subject are identified and adjusted for a reference input, it then predicts mean eye tracking behavior for that subject for inputs of comparable complexity.


On the perception of some geometric properties of rotating three dimensional objects.

Various observations and conjectures have been made about how we perceive three dimensional objects under monocular viewing conditions; in particular, when such objects are rotating. In this paper these proposals are reviewed and reinterpreted in the light of geometric properties of objects and their projections. Some results are also presented which support the conclusion that the visual system, in reconstructing the object from its projections, requires a fixed time to establish the objects curvature and torsion parameters. In the perceptual reconstruction process these parameters assume positive or negative values, and this equivocation is related to a form of the adjacency principle involving central projections and "perceptual geodesics".


The response of a spatially distributed neuron to white noise current injection.

The depolarization of passive nerve cylinder or dendritic tree in the equivalent cylinder representation is assumed to satisfy the cable equation. We consider in detail the effects of white noise current injection at a given location for the case of sealed end boundary conditions and for an initial resting state. The depolarization at a point is a Gaussian random process but is not Markovian. Expression (infinite series) are obtained for the expectation, variance, spatial and temporal covariances of the depolarization. We examine the steady state expectation and variance and investigate how these are approached in time over the whole neuronal surface. We consider the relative contributions of various terms in the series for the expectation and variance of the depolarization at x = 0 (soma, trigger zone, recording electrode) for various positions of the input process. It is found that different numbers of terms must be taken to obtain a reasonable approximation depending on whether the stimulus is at proximal, central or distal parts of the dendritic tree. We consider briefly the interspike time problem and see in an approximate way how spatial effects are important in determining the mean time between impulses.


[Lipid regulation of carbohydrate transport system in Mycoplasma].

Cultivation of Acholeplasma laidlawii cells in media containing unsaturated fatty acids results in changes of the physiological state of the membrane lipid bilayer due to preferable incorporation of an unsaturated fatty acid into lipids. The lipids are capable to regulate the transport activity since the transport rates for glucose, 3-O-methyl-C-glucose, glucerol and erythritol change considerably when the cells are cultivated in media containing different unsaturated fatty acids. The transport activity is also affected by the length of the carbon chain, the degree of the fatty acid saturation and the presence of cholesterol. At the same time the activation energy of the transport activity also changes, which suggests that the regulation by lipids (presumably local changes of the physical properties of lipid domen) is involved in the process of the carrier association with the substrate and/or in translocation of this complex through the membrane.


[Proteinases in growth and spore formation of Bacillus thuringiensis].

Some serine proteases and leucine aminopeptidases were detected inside and outside the cells during the analysis of three crystalline and two acrystalline strains of Bac. thuringiensis var. galleriae. The data obtained on the protease formation during growth and sporulation and the level of their activity are indicative of intracellular proteases involvement in spore- and crystal formation. The enzymes isolated from the culture medium do not probably take part in these processes. The intracellular enzymes may account for the different crystal protein composition of various strains due to limited proteolysis of crystal proteins in the course of biosynthesis.


[Bacterial membrane proteins. Properties of Micrococcus lysodeikticus NADH dehydrogenase].

NADH dehydrogenase was isolated from M. lysodeikticus membranes with FAD as a prosthetic group. It was found the enzyme molecular weight is about 140000 in 0,01 M phosphate buffer, pH 7,4 in 1% Triton X-100. The enzyme molecules are dimers consisting of two subunits with molecular weight of 70000. The content of alpha-helical regions is 30%, that of beta-forms is 13%. The protein globule is cross-linked with the disulfide bonds and has hydrophobic regions on its surface.


[Primary structure of abnormal E-like hemoglobin].

An E-like abnormal hemoglobin was detected in a hematological patient and one of the members of her family. The composition of blood hemolyzates was characterized using acetate cellulose electrophoresis. The abnormal beta-chain was isolated by ion-exchange chromatography of total globin on CM-cellulose. Using peptide mapping of the abnormal beta-chain trypsin hydrolysates, it was shown that the amino acid substitution occurs in peptide beta T3. The amino acid analysis and determination of the abnormal fragment C-terminal amino acid allowed to establish the locus and type of this substitution. The first case of hemoglobin E(alpha 2 beta 2 26Glu leads to Lys) identification on the territory of the USSR is reported.


[Inhibition of succinate and NADH oxidases of submitochondrial particles by iron chelators and sulfhydryl reagents].

The inhibition of succinate- and NADH-oxidase activities of submitochondrial particles by 4,7-diphenyl-1,10-phenantroline was studied. The inhibition was shown to increase when the particles were pretreated with SH-reagents. The treatment of submitochondrial particles with ethanol in the presence of 1,10-phenantroline resulted in a complete inactivation of succinate oxidase and succinate: tetramethyl-n-phenyldiamine reductase; the succinate PMS reductase activity was only partially inhibited after such treatment. It is concluded that tetramethyl-n-phenyldiamine and phenazine metasulfate react with different sites of the succinate dehydrogenase complex. The changes in the properties of submitochondrial particles after ethanol--phenantroline treatment are apparently due to the effect of non-polar solvent rather than to the extraction of non-haem iron.


[Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver].

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.


[Changes in DNA methylation in alfalfa plants infected with Cuscuta and tissue differences in DNA methylation of the parasite plants].

The tissue-specific differences in the 5-methylcytosine (m5C) content in total DNA of the parasite plant Cuscuta reflexa have been found: DNA from apical parts of the plant is less methylated (m5C = 4,2 mol %) as compared to the DNA from haustoria and posthaustorial regions (m5C = 5,4 mol %). The base compositions of total DNA preparations from C. reflexa grown on various hosts are similar. The m5C amount in stem DNA of the alfalfa plant infected with C. reflexa is by approximately 25% higher than that in the non-infected plant DNA. The GC content in alfalfa DNA does not change as a result of infection. Thus, the parasite induces the hypermethylation of DNA in the host plant. It is assumed that the changes in DNA methylation induced by the parasite plant may play a regulatory role and may cause changes in transcription and replication of host DNA.


[Mechanisms of liver transketolase activation in B1-deficient rats after administration of thiamine].

Gel filtration and equilibrium dialysis demonstrated that the hyaloplasmic fraction of the liver of B1-deficient rats does not practically bind C-TDP in vitro. An addition of the excess of non-labelled coenzyme does not increase the transketolase activity. The data obtained suggest that transketolase activation in the hyaloplasmic fraction of the liver of B1-deficient rats after administration of thiamine in vivo is due to stimulation of the additional synthesis of the enzyme protein rather than to the saturation of the free apoenzyme with newly-formed TDP. In vivo and in vitro studies suggest that the hyaloplasmic fraction of the liver of B1-deficient rats contains no free apoenzyme of transketolase.


[Effects of organic solvent vapors on the protochlorophyllic complex from etiolated leaves. Conditions for reversible and irreversible destruction].

The spectral properties and the ability of etyolated leaves pigments treated with organic solvent vapours (OS) for phototransformations were studied by measuring low temperature fluorescence spectra (-196 degrees C). Under the effects of OS the fluorescence at 655 nm was gradually decreased and that at 630--640 nm was increased. The effects of OS depended on the partial pressure of OS. The ability of the pigments for phototransformations was decreased with an increase in fluorescence at 630 nm. The emission maximum of fluorescence of the pigment formed in the light was shifted by 10--18 nm towards the shortwave region. Partial reversibility of the destroying effects of diethyl ester was found. A removal of the ester vapours resulted in a relative increase of fluorescence in the etyolated leaves at 640--645 nm and a decrease of the amount of "photo-inactive" pigment. The maximum of fluorescence of the pigment formed in the light was shifted towards the long-wave region (approximately 5 nm) as compared to the leaves irradiated in the presence of the ester. Partial functional reconstitution indicates that at least part of the pigment molecules are able to form a protochlorophyllide (protochlorophyll) -- protein complex, similar to the native one.


[Activities of dehydrogenases of the pentose phosphate pathway and transketolase in transplanted mouse hepatomas with different growth rates and in organs of tumor carriers].

The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transketolase were studied in the cytoplasmic fractions of transplanted mouse hepatomas differing in their growth rates, and in the liver, spleen and cortical layer of kidneys of tumour carriers and normal mice. It was shown that transplantation of hepatomas changes the activity of the pentose phosphate pathway enzymes in tumour carrier tissues unaffected by neoplasm. Deviations from normalcy were mainly similar to those observed in the hepatomas. The changes in the enzymatic activities were especially well-pronounced in the mice having rapidly growing hepatomas. This may be due to a generalized effect of the tumour on the organism, which is concurrent with malignancy.


[Determination of structural differences in DNA by pyrimidine isopliths ratios].

Using a modified Burton procedure, twelve pyrimidine nucleotide isopliths of DNA from five mammalian species (human, rabbit, guinea pig, rat and cattle) were determined. A method is proposed for mathematical estimation of DNA block analysis data, revealing a correlation between the specific DNA primary structure, the systemic status of the organism under investigation and the organism's radiosensitivity. In some cases DNA structural differences as determined by pyrimidine isoplith ratios help to distinguish between families of the same mammalian order. Quantitative isoplith ratios demonstrate that ionizing radiation treatment brings about certain changes in DNA primary structure. Their direction is quite the opposite to the main trend in the changes of DNA structure in the course of biological evolution.


[Effects of alpha-tocopherol derivatives on natural quinone levels in the tissues of vitamin E deficient rats].

The effects of alpha-tocopherol and its derivatives (alpha-tocopherylquinone, its short-chained analog--alpha-tocopheronolactone--and short-chained alpha-tocopherylacetate) on the levels of ubiquinone, its cyclic isomer--ubichromenol--and vitamin E in the liver and heart of vitamin E-deficient rats were studied. After injection of alpha-tocopherol derivatives the levels of ubichromenol and ubiquinone in rat liver and heart were increased, while their ratio was decreased. alpha-Tocopheronolactone was found to exert the strongest action, which is probably due to its direct effect on ubiquinone metabolism in rat tissues.


[Lateral heterogeneity of bacterial membranes].

Isolated membranes of M. lysodeikticus were rapidly frozen and disrupted in a Hughes press. After disruption the fragments were centrifuged at 144000 g for 1 hour and part of the supernatant just above the pellet was subjected to isopycnic centrifugation in a continuous sucrose density gradient. It was found that the material tested was a mixture of fragments differing in their buoyant densities. These fragments also differed in their protein/lipid ratios, cytochrome content and dehydrogenase activities calculated per protein and lipid as well as in proportion of the respiratory chain enzymes. The results obtained are indicative of lateral heterogeneity of the bacterial membrane and the existence of areas in the membrane having high concentration of the respiratory chain enzymes. The latter may suggest that the system of substrate oxidation is segregated in the membrane. It is assumed that there exists in the membrane an exchange of components between different electron-transporting chains operated due to their lateral diffusion.


[Isolation and properties of glucose-6-phosphate dehydrogenase from human cultivated cells].

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.