Interactions of La3+ with phosphatidylserine vesicles. Binding, phase transition, leakage and fusion.

The interaction of La3+ with phosphatidylserine vesicles is elucidated by binding studies, differential scanning calorimetry, X-ray diffraction, freeze fracture electron microscopy, and release of vesicle contents. La3+ effectively competes with Ca2+ for phosphatidylserine binding sites. The saturation level is close to a La/lipid ratio of 1:3. A concentration of 0.1 mM of La3+ is sufficient to induce fusion between sonicated vesicles.


Selective decoration of hydrophilic moieties of membrane molecules in freeze fracture.

Selective decoration of the hydrophilic moieties of phospholipid molecules on freeze fractured bilayer faces was made using residue water vapor in an oil-free vacuum unit. The preferential decoration technique was applied to label structural faults of bilayers, such as domain boundaries and other regions of molecular dislocation which are not visible by conventional morphological observations.


The maxi-circle of Trypanosoma brucei kinetoplast DNA.

We have constructed a fragment map of the maxi-circle component from kinetoplast DNA networks of Trypanosoma brucei brucei (East African Trypanosomiasis Research Organization strain No. 427), using restriction endonucleases PstI, HapII, EcoRI, BglI, HindIII, HhaI, SstI, XbaI, HaeIII and Sau-96I. Although the 20 kilo-base pair map contains 30 fragments, there is a 6.5 kilo-base pair 'silent' segment which lacks recognition sites for any of these enzymes. In CsCl intact kinetoplast DNA has a buoyant density of 1.690 g/cm3, whereas a kinetoplast DNA fraction enriched in maxi-circle sequences contains an additional component at 1.682 g/cm3. The possibility that the 'silent' segment is very rich in A + T is discussed.


Precursor-product relationships between thymidine nucleotides and DNA in mammalian cells. II. Studies with dTDP and dTTP on cells partially lysed by Brij-58.

Cells from Chinese hamster ovary cell cultures were partially lysed with Brij-58 and incubated in a reaction mixture containing deoxyribonucleoside triphosphates and permitting semiconservative DNA replication. The reaction mixture was supplemented with [3H]dTTP and [14C]dTDP or, alternatively, with [14C]dTTP and [3H]dTDP, and at different times, 3H and 14C specific activities of dTTP and dTDP, as well as 3H/14C ratios in DNA were determined. Isotope ratios in DNA were in good agreement with those expected if dTTP but not dTDP was used as the proximate precursor for DNA synthesis. These data support the conclusion that the thymidine nucleotide used as proximate substrate for DNA replication is dTTP.


Hydroxyapatite chromatography of short single-stranded DNA.

Short single-stranded segments of calf thymus DNA were obtained by random cleavage with DNAase I. After treatment with various concentrations of DNAase I, fragment sizes were estimated using the ratio of total to terminal phosphorus. DNA populations ranging from 4--180 bases were obtained. Fragments with lengths up to 1140 were generated by shearing in a Virtis homogenizer. The hydroxyapatite elution profiles of sized populations were determined by elution with phosphate gradients. A curve relating elution molarities to single-strand chain length was 'biphasic', with the elution molarity being extremely sensitive to chain lengths below 50 nucleotides but much less sensitive to chain lengths above 100 nucleotides. These results show that single-stranded fragments below 50 nucleotides elute from hydroxyapatite appreciably before high molecular-weight denatured DNA using phosphate gradients. This is an important consideration when using hydroxyapatite to fractionate DNA populations which contain short single strands.


Direct in vitro photoaffinity labeling of DNA with daunorubicin, adriamycin, and rubidazone.

Irradiation by daylight fluorescent lamps, of a physical complex of duplex DNA with either daunorubicin, adriamycin, or rubidazone produced covalent adducts to DNA. The photoincorporated drug could not be extracted by phenol extraction which removed 99% of the non-photolyzed drug from its physical complex with DNA. The photoadduct was also stable to dialysis, Mg2+ addition, column chromatography, and thermal denaturation and alkali treatment of the DNA. The photoinduced adduct was proportional to the amount of incident irradiation, and the amount of DNA present, and as much as 30--45% of the drug which was physically associated could be photoincorporated. The drugs were not incorporated extensively into single-stranded DNA which lacks the ability to bind these antitumor agents by intercalation. Although the photochemical mechanisms of photoaffinity labeling DNA with these antineoplastic agents are unknown, this approach may prove to be useful for trageting their cellular sites of actions.


Stability of bacterial messenger RNA in mesophiles and thermophiles.

The decay of [3H]uridine-labeled mRNA was measured in the mesophile, Bacillus licheniformis (grown at 37 degrees C and 46 degrees C), and in the thermophile, Bacillus stearothermophilus (grown at 46 degrees C and 55 degrees C). For each organism, the half-life of the mRNA decreased as the growth temperature was increased. The stability index (half-life of mRNA/doubling time of cells), however, was remarkably constant for each organism regardless of the growth temperature. It is concluded that these results support the concept that kinetic considerations play a significant role in the explanation of thermophily.


Fate of mRNA following disaggregation of brain polysomes after administration of (+)-lysergic acid diethylamide in vivo.

Intravenous injection of (+)-lysergic acid diethylamide into young rabbits induced a transient brain-specific disaggregation of polysomes to monosomes. Investigation of the fate of mRNA revealed that brain poly(A+)mRNA was conserved. In particular, mRNA coding for brain-specific S100 protein was not degraded, nor was it released into free ribonucleoprotein particles. Following the (+)-lysergic acid diethylamide-induced disaggregation of polysomes, mRNA shifted from polysomes and accumulated on monosomes. Formation of a blocked monosome complex, which contained intact mRNA and 40-S plus 60-S ribosomal subunits but lacked nascent peptide chains, suggested that (+)-lysergic acid diethylamide inhibited brain protein synthesis at a specific stage of late initiation or early elongation.


Enzymatic preparation of adenosine 5'-O-(3-[35S]thiotriphosphate) by thiophosphorylation of adenosine diphosphate.

The very useful radiolabeled ATP analog, adenosine 5-O-(3-[35S] thiotriphosphate) or [35S] ATPgammaS, has been prepared by a technique based on the thiophosphorylation of ADP that results in much higher yields of [35S] ATPgammaS than does a thiophosphate exchange method [1].


A physical map of bovine mitochondrial DNA from a single animal.

Mitochondrial DNA has been isolated from the liver of an individual Holstein cow and a physical map has been derived for the 38 cleavage sites made by restriction endonucleases: Ava I, Bam HI, Bgl II, Bst EII, Eco RI, Hha I, Hin dIII, Hpa I, Kpn I, Pst I, Sac I, Sal I, Xba I, and Xho I. Sufficient mitochondrial DNA (approx. 16 mg) could be isolated, allowing this map to serve as the basis for detailed physical, genetic and nucleotide sequence studies in an individual mammal.


Characterization of long and short repetitive sequences in the sea urchin genome.

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.


The secondary structure of bacteriophage DNA in situ. VIII. The reaction of sodium bisulphite with intraphage cytosine as a probe for studying the DNA-protein interaction.

To obtain data on the viral nucleoprotein a study has been made of the reaction of sodium bisulphite with cytosine in the intraphage DNA of the phage Sd. The CHlO4 hydrolysates of the bisulphite-modified phage Sd have demonstrated a decrease of 18% in the cytosine content and the presence of the products with the properties of cytosyl-amino acids (the main amino acid responsible for the DNA-protein interaction involving cytosine is lysine). But when prior to hydrolysis the modified phage was disintegrated under mild conditions in 0.1--1 M NaCl solution or Tris-HCl buffer (pH 7), neither the decrease in the cytosine content nor cytosyl-amino acids have been found. An exception is the heating of the phage at 70 degrees C in a medium containing 0.05 M phosphate buffer (pH 7.9--8.5), when an 18% decrease in the cytosine content and subsequent appearance of cytosyl-amino acids have also been observed. The presence of cytosyl-amino acids which are the nucleotide-protein cross-links is confirmed by the results of viscometry, equilibrium centrifugation in cesium sulphate gradient and determinations of the survival percentage. It is suggested that the reaction between bisulphite and cytosine in the phage Sd stops at the stage of the intermediate product C5-C6-dihydro-C6-sulphopyrimidine whose amino group is shielded by interaction with protein (product VII). This product can exist only under in situ conditions: with disintegration of nucleoprotein (destruction of phage particles or ejection of the DNA) in phosphate-free media the product VII reverts into the initial cytosine. Under the conditions of acid hydrolysis or destruction of phage in the presence of phosphate ions product VII undergoes transamination with cleavage of SO3 and restoration of the C5-C6 double bond producing cytosyl-amino acids. The factors determining the stability of the product VII are discussed.


Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene.

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.


Partial purification of deoxyuridine triphosphate nucleotidohydrolase and its effect on DNA synthesis in isolated HeLa cell nuclei.

Deoxyuridine triphosphate nucleotidohydrolase (EC 3.6.U.23) has been partially purified from HeLa S3 cells, and found to have an apparent molecular weight of 50--55 000 by gel filtration under non-denaturing conditions. The enzyme is specific for the hydrolysis of dUTP, requires Mg2+ and is inhibited by EDTA. The apparent Km for dUTP is 0.1 microM. Isolated HeLa cell nuclei were treated with dUTPase before pulse-labelling with [3H]dTTP which also had been pretreated with dUTPase. This pretreatment changed neither the total amount nor the size of the primary DNA pieces. A role for dUTP incorporation in their genesis can therefore be excluded and these primary DNA pieces are considered to be true intermediates in discontinuous DNA replication.


1,3-beta-D-glucanases from Pisum sativum seedlings. II. Substrate specificities and enzymic action patterns.

Two purified pea 1,3-beta-D-glucanases (EC 3.2.1.6) hydrolyse laminarin (degree of polymerization 20), laminaridextrins (degree of polymerization 3--7), and their reduced 3H-derivatives, 1,3-beta-D-glucans which are partially substituted (carboxymethyl-pachyman) or crystalline (curdlan), and mixed-linkage beta-glucans. Enzyme kinetics and product-formation indicate endo-hydrolase activity with weak transglycosylase capacity. The enzymes do not hydrolyse beta-glucosides, the 1,3 linkage adjacent to the reducing end of chains, or cellulose and its derivatives. They degrade mixed-linkage beta-glucans, in a manner similar to Rhizopus arrhizus endo-1,3-beta-D-glucanase, to form the products expected from hydrolysis of linkages adjacent to 1,3-beta linkages. With respect to action patterns, glucanase I (from apical growing tissue) differs from glucanase II (from basal maturing tissue) in several respects: (a) on a molar basis, I generates reducing groups from all substances more rapidly than II; (b) lower laminaridextrins are hydrolysed by I at the non-reducing terminal linkage, while II preferentially hydrolyses internal linkages; (c) laminarin is hydrolysed to lower laminaridextrins by I more rapidly than II, but I takes longer than II to completely degrade laminarin chains; (d) the enzymes are differentially sensitive to different classes of non-competitive inhibitors. It is concluded that these beta-glucanases differ in such a way that I preferentially continues to degrade fragments produced by endo-hydrolytic attack on long chains ('multiple attack' action pattern), while II hydrolyses internal linkages of the longest chains available ('multi-chain attack').


A latent gelatin specific proteinase of human leucocytes and its activation.

1. Gelatin specific proteinase (gelatinase) exists in human leucocytes extracts mainly in a latent form. 2. It is activated by different proteinases as well as by some chemicals (urea, NaSCN, HgCl2). 3. Non-proteolytic activation of latent gelatinase and the decreasing of its molecular weight associated with it strongly suggests that it is an enzyme-inhibitor complex.


Comparative studies on the mechanism of activation of the two human trypsinogens.

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.


Biphasic kinetics of metal ion reactivation of trypsin-thiol complexes.

This report describes biphasic kinetic data obtained when trypsin was inhibited by a thiol-containing inhibitor present in Ehrlich ascites tumour cells and then subjected to addition of Hg2+, Cu2+ or Ag+. This resulted in an initial re-activation of the trypsin, followed by inhibition of the enzyme with the addition of higher concentrations of these ions. The significance of these observations is 2-fold: (i) help to elucidate the mechanism of metal ion activation of latent enzymes, and (ii) also indicate that, in certain circumstances, the concentration of added metal ion determines whether the metal acts as an activator or an inhibitor of enzyme activity.


An enzyme-polymer film prepared with the use of poly(vinyl alcohol) bearing photosensitive aromatic azido groups.

Photochemical reaction of poly(vinyl alcohol) bearing aromatic azido groups was applied for immobilization of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21.) in poly(vinyl alcohol) film. Photo-crosslinking and immobilization reactions proceeded by light irradiation for 25 min in air. The immobilized enzyme showed approx. 40% of its native enzyme activity with an apparent Michaelis constant of 3.9 mM. The Michaelis constant of the native enzyme was 2.3 mM. Some properties of the immobilized and native enzyme are compared.


The stereoconfiguration of newly formed molecules of bis(monoacylglycero)phosphate in BHK cells.

Newly formed molecules of bis(monoacylglycero)phosphate (known also as lysobisphosphatidic acid), which were labeled with 32Pi in cultured BHK cells during relatively short pulses, were subjected to stereoanalysis. In contrast to the high proportion of sn-1-glycerophosphate residues in the bulk of the bis(monoacylglycero)phosphate molecules, the newly formed molecules were rich in sn-3-glycerophosphate residues.


A selective deficiency of hepatic triacylglycerol lipase in guinea pigs.

The properties of postheparin plasma triacylglycerol-hydrolyzing enzymes were investigated in guinea pig and rat. In rat, lipoprotein lipase and hepatic triacylglycerol lipase were separated on a heparin-Sepharose affinity chromatography. In postheparin plasma of guinea pig, however, hepatic triacylglycerol lipase was almost completely absent, while lipoprotein lipase was present. Hepatic triacylglycerol lipase was also deficient in the liver tissue extract of guinea pig. Plasma lipoprotein compositions of high-fat fed and control guinea pigs were analyzed. One of the outstanding changes found in high-fat fed animals was the presence of chylomicronemia. One guinea pig showed gross hyperlipemia with triacylglycerol concentrations of 2715 mg/100 ml. Plasma triacylglycerol concentrations of each lipoprotein fraction of very low density, intermediate density, low density and high density lipoproteins from high-fat fed animals were almost the same as those of the corresponding lipoprotein fractions from controls. Discussion was focused on the development of chylomicronemia in relation to the defects of triacylglycerol-hydrolyzing enzyme systems in this animal.


Synthesis of various kinds of esters by four microbial lipases.

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.


Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate.

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.


Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques.

1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. Microsomal lysophospholipase activity was completely inhibited by [3H]diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrohporesis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [125I]iodosulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.


Comparison of rates of hydrolysis of N-oleoyl and N-stearoyl glucocerebroside in patients with Gaucher's disease.

The deficiency of oleic acid as one of the fatty acids in glucocerebrosides that accumulate (31--77 mg/g dry weight) in the spleen in patients with Gaucher's disease was confirmed in 9 cases. In an effort to account for the 10-fold difference between the oleoyl glycocerebroside content of glucocerebrosides in spleen from controls and patients with Gaucher's disease, we compared the ability of extracts of spleen and fibroblasts from individuals with various forms of Gaucher's disease and controls to hydrolyze [14C]stearoyl and [3H]oleoyl glucocerebroside. The residual glucosylceramidase activity in patients with Gaucher's disease hydrolyzes the glucose moiety of oleoyl glucocerebroside at approximately the same rate as that of stearoyl glucocerebroside. Similarly, the more active glucosylceramidase of control tissue acts upon both oleoyl and stearoyl glucocerebrosides with equal efficiency. These observations indicate that a mutation affecting the substrate specificity of glucosylceramidase cannot account for the lack of oleic acid-containing glucocerebrosides in patients with Gaucher's disease. Thus, the hypothesis that the difference in fatty acid composition found in glucocerebroside is obtained as a result of a mutation affecting the specificity of the residual glucosylceramidase must be rejected.


Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats.

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.


Dietary regulation of 3-hydroxy-3-methylglutaryl-CoA reductase from rat intestine.

The effect of dietary cholesterol on rat intestinal 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) varied depending upon whether animals received the dietary cholesterol with polyunsaturated or saturated fats. When cholesterol was fed with polyunsaturates, the enzyme activity in both the jejunum and ileum was significantly suppressed, whereas only the enzyme in the jejunum was significantly suppressed when cholesterol was given with saturated fats. It is concluded that dietary cholesterol has a negative feedback effect on intestinal cholesterol synthesis.


Exchange of methyl hydrogens in ethanol during incorporation in bile acids in vivo.

[2,2,2-2H]Ethanol was administered continuously to bile fistula rats for 72 h, with or without (--)-hydroxycitrate. The deuterium labelling of biliary bile acids was determined by GC-MS and 13C NMR. Difference spectra between 2H,1H- and 1H-decoupled 13C NMR spectra showed the presence of partly deuterated methyl and methylene groups in methyl cholate, indicating exchange of deuterium in [2,2,2-2H]ethanol for protium prior to or during incorporation of acetate into the bile acid. The extent of exchange was 20--30% as calculated from the isotopic composition of a fragment ion containing one methyl and one methylene group derived from C-2 of acetate. The exchange was unaffected by (--)-hydroxycitrate, indicating that it was not due to reversible incorporation of deuterated acetate into citrate. About 60% of the acetyl-CoA serving as precursor of cholic and chenodeoxycholic acids were derived from ethanol. This value was not changed by administration of (--)-hydroxycitrate. The half-life time of cholesterol molecules acting as precursors of both bile acids was about 50 h in the presence of (--)-hydroxycitrate, which is about the same as previously found in the absence of the inhibitor.


Regulation of ether lipids and their precursors in relation to glycolysis in cultured neoplastic cells.

Tumors typically show high rates of glycolysis and elevated levels of ether lipids, particularly the alkyldiacylglycerols; thus, we investigated the relationship between ether lipid accumulation and glucose metabolism in a neoplastic cell line (B2-1). The B2-1 cells grown in 5.5 mM galactose in the absence of glucose produced very low levels of alkyldiacylglycerols, triacylglycerols, lactic acid, and dihydroxyacetone-P. Increasing concentrations of glucose caused a progressive increase in lactic acid, dihydroxyacetone-P, and up to a ten-fold increase in alkyldiacylglycerols and triacylglycerols. Glucose supplements also caused an increased incorporation of [9,10-3H]palmitic acid into alkyldiacylglycerols and triacylglycerols. These metabolic changes appeared to be independent of altered growth rates of the cells. The addition of hexadecanol along with glucose to the cultures resulted in a shorter lag and a more rapid rate of accumulation of alkyldiacylglycerols; hexadecanol supplements alone had no effect. The extent of uptake and oxidation of hexadecanol was similar in both the glucose and galactose-grown cells. These results indicate that the levels of alkyldiacylglycerols in neoplastic cells can be regulated by the extent their precursors are formed from glucose.


Polar lipids of Pseudomonas vesicularis. Presence of a heptosyldiacylglycerol.

The individual polar lipids produced by Psuedomonas vesicularis NCTC 10 900 during surface culture have been isolated. The major lipids are phosphatidylglycerol, a phosphatidyl-alpha-D-glucopyranosyldiacylglycerol, 1,2-di-O-acyl-3-O-alpha-D-glucopyranosylglycerol. 1,2-di-O-acyl-3-O-alpha-D-glucopyranuronosylglycerol, and 1,2-di-O-acyl-3-O[beta-D-glucopyranosyl-(1 yields 4)-alpha-D-glucopyranuronosyl]glycerol. These are also the major polar lipids of Pseudomonas diminuta. Additional lipids present in P. vesicularis are unidentified carotenoids and a novel lipid characterised as 1,2-di-O-acyl-3-O-alpha-D-glycero-D-glucoheptopyranosylglycerol. A cis-octadecenoic acid and hexadecanoic acid are the major fatty acids: C15 and C17 acids are significant minor components. The fatty methyl ester fractions derived from three of the lipids (most notably the glucosyldiacylglycerol) contained substantial amounts of a compound with chromatographic properties resembling those of an octadecenoic ester: the identity and origin or this compound remained uncertain.


Effects of various oxygenated sterols on cellular sterol biosynthesis in Chinese hamster lung cells resistant to 25-hydroxycholesterol.

The effects of a wide variety of oxygenated sterols upon sterol biosynthesis and hydroxymethylglutaryl-CoA reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) activity in a wild-type clone and in a 25-hydroxycholesterol-resistant clone of Chinese hamster lung (Dede) cells are described. Derivatives of cholesterol which were oxygenated in the 6, 7 or 15 positions of the sterol nucleus or in the 20, 22, 24 or 25 positions of the sterol side chain were shown to be potent inhibitors of sterol synthesis and reductase activity in the wild-type cells but none of these substitutions had any effect on the 25-hydroxycholesterol-resistant A2 clone. A 32-hydroxylated derivative of lanosterol also suppressed sterol synthesis and reductase activity in wild-type cells but had no significant effect upon the A2 line. It was also appraent that a complete sterol side chain was necessary for inhibitory activity. Studies of a wide range of inhibitory sterols indicated that there was a close correlation between their effects upon sterol synthesis and reductase activity and that their inhibitory action was specific for sterol biosynthesis since little effect was observed upon fatty acid or CO2 synthesis. Previous studies had shown that the uptake of 25-hydroxycholesterol by the resistant A2 line was unimpaired and the present results indicate that metabolism of this oxygenated sterol is also unaltered. These results, in conjunction with previous studies, suggest that the resistant A2 line is defective in feedback regulation of cholesterol synthesis and that all of the oxygenated sterols tested suppress the biosynthetic pathway through at least one common step.


Effects of ML-236B on cholesterol metabolism in mice and rats: lack of hypocholesterolemic activity in normal animals.

ML-263B (compactin), a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, is very effective in lowering serum cholesterol levels in animal species such as hens, dogs, monkeys and man. In the present studies, the effect of this drug on cholesterol metabolism in several strains of mice and rats was studied. The results indicate that, when administered for a longer period, the drug showed no hypocholesterolemic activity in these species under either normo- or hypercholesterolemic conditions, except for rats treated with the detergent Triton WR-1339. The administration of ML-236B caused a significant decrease in fecal excretion of bile acids and in the hepatic levels of cholesterol 7 alpha-hydroxylase, and produced a marked increase in hepatic levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity, resulting in no inhibition of hepatic sterol synthesis, even in the presence of the drug in the active form(s). It is concluded that the lack of hypocholesterolemic activity of ML-236B in mice and rats could, at least partly, be explained by these unexpected results.


The structure of canine intestinal trihexosylceramide.

One of the neutral glycosphingolipids isolated from dog intestine has a mobility on thin-layer chromatography and a carbohydrate composition similar to trihexosylceramides. Structural analysis has shown that it consists largely of isoglobotriaosylceramide, galactosyl(alpha-1-3)galactosyl(beta-1-4)glucosyl(beta 1-1')ceramide.


Effect of alcohols on the collagen-phosphatidylcholine interaction.

The interaction of phosphatidylcholine dispersions with acid soluble collagen separated from the skin of one month-old swine was studied to define the conditions facilitating the association of the collagen with lipids. When acid soluble collagen and phosphatidylcholine dispersions were incubated in 75 mM citrate buffer of pH 3.7 at 25 degrees C, the reisolated collagen fibrils did not contain appreciable amounts of phosphatidylcholine. However, the presence of n-propanol greatly promoted the retention of phosphatidylcholine, the amount of phosphatidylcholine associated being nearly 30% of collagen on a weight basis under optimal conditions. In contrast, methanol, ethanol, isopropanol, and n-butanol did not appreciably enhance the association of phosphatidylcholine with collagen. A limited inhibition of phosphatidylcholine retention was observed upon addition of sodium chloride to the propanol medium. The interaction of phosphatidylcholine with acid soluble collagen decreased sharply when temperature was increased above 30 degrees C; almost no phosphatidylcholine-collagen association occured at 40 degrees C. It appears that the enhanced association in the presence of n-propanol is due to a looseing of the collagen triple helix that exposes hydrophobic sites necessary for the interaction. However, the conversion of the triple helical structure to the random coil conformation by heating prevents the association of phosphatidylcholine with acid soluble collagen.


The presence of citrulline in epidermal proteins.

Citrulline is present in the stratum corneum proteins of human, cow snout, pig snout and guinea pig epidermis but is absent from the stratum corneum proteins of frog, mouse, turtle, rat and hamster epidermis. The amino acid is released by acid hydrolysis and ranges from 1.7 to 5.5 residues per thousand residues of protein amino acid. Protein derived citrulline co-chromatographs with authentic L-citrulline on an amino acid analyzer, on Dowex-50, on Dowex-2 and on thin-layer chromatography. Dansylated material co-chromatographed with authentic dansyl-L-citrulline in two thin-layer chromatography systems. Labelling experiments have shown that the protein bound citrulline is derived from protein bound arginine and probably results from enzymatic conversion of the guanido group to the ureido group.


Polypeptides of the synaptic membrane antigens D1, D2, and D3.

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.


Isolation of an acidic protein from the armadillo submandibular gland.

An acidic protein fraction with an apparent molecular weight of 34 000 has been isolated from the Cetavlon-treated, mucin-free supernatant of the armadillo submandibular gland 0.01 M NaCl extract. This purified material, which was obtained in a yield of 0.45%/g wet gland, contains 24 mol % acidic amino acids and 4 mol % basic amino acids. Hexosamines, sialic acid, and neutral sugars represent 7% of the dry sample weight. In polyacrylamide gel and cellulose acetate electrophoresis, a single protein band was observed. The acidic protein fraction is highly reactive with the Lowry phenol reagent, giving a protein value 83% higher than that obtained by summation of its anhydrous amino acids, and is explained by the occurrence of peptide linkages peculiar to this material. The presence of other basophilic components besides mucus glycoproteins within the salivary gland of the armadillo may have physiological significance.


Alteration in tryptic peptide patterns of ferritins purified from human colon carcinoma.

Ferritin from malignant tissue differs electrophoretically from normal ferritin. The molecular basis of this difference has not yet been defined. Malignant tissue contains a mixture of ferritins from normal cells, inflammatory cells as well as cancer cells. GW-39 is a pure colon carcinoma cell system that synthesizes human carcinoembryonic antigen. Therefore, ferritin was isolated from normal colon mucosa and colon cancer tissues, as well as from the colon carcinoma cell line, to clarify the molecular relationship between normal and malignant ferritins. Colon carcinoma ferritin differs in primary structure from normal colon mucosal ferritin and contains at least six additional different tryptic peptides. These six peptides were also found in the ferritin from the colon carcinoma cell line. These data suggest that the alteration in ferritin structure occurs at the cellular level and is associated with the malignant state.


Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin.

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.


Purification and characterization of a lectin from seeds of the winged bean, Psophocarpus tetragonolobus (L.)DC.

A lectin from seeds of the winged bean, Psophocarpus tetragonolobus (L.)DC, has been purified to homogeneity by affinity chromatography on a Bio-Gel lactobionate adsorbent. The lectin is rich in acidic amino acids, and after isoelectric focusing in polyacrylamide gel, appeared as a single band of pI5.5 The molecular weight of the lectin is 46 000 +/- 2000 as determined by analytical ultracentrifugation. Dodecyl sulfate gel electrophoresis detected a single type of subunit with a molecular weight of 29 000 +/- 3000. The lectin has specificity for receptors containing D-galactose and agglutinates trypsinized human red blood cells of all three ABO types. Of the tested inhibitors of hemagglutination caused by winged bean lectin, p-nitrophenyl-beta-D-galactoside was the most potent. Lactose was the most active oligosaccharide tested, and other oligosaccharides containing alpha-linked D-galactose were much less inhibitory.


Characterization of a membrane protein from cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata.

Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.


Purification of bovine adrenocortical and brain tubulin. A comparative study.

Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue.


Evidence for a structural role for chlorophyll in chlorophyll-protein complexes.

1. Chymotrypsin treatment of spinach chloroplast membranes does not change the electrophoretic mobility of either chlorophyll-protein complex 1 or 2. 2. The extraction of lipids with 80% acetone after treatment of the membranes with chymotrypsin reveals that the polypeptide components of both chlorophyll-protein complexes had been extensively digested. The extraction of carotenes with petroleum ether under the same conditions does not change the electrophoretic mobility of the chlorophyll-protein complexes. 3. Fluorescence polarisation studies of chlorophyll-protein complex 2 reveal that the chymotrypsin digestion of this complex does not result in changes of mutual orientation or distance apart of chlorophyll a, chlorophyll b or carotenoid. 4. Two polypeptide components have been detected after lipid extraction of electrophoretically purified chlorophyll-protein complexes 1 and 2. The SDS molecular weights are 24 000 and 27 000 for complex 2, and 68 000 and 64 000 for complex 1. 5. We conclude that chlorophyll performs an important structural function in both chlorophyll-protein complexes.


Bovine type I collagen: A study of cross-linking in various mature tissues.

The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000.


The anti-proteolytic behavior of lathyrogens.

The lathyrogens, beta-aminopropionitrile and alpha-aminoacetonitrile inhibit both the esterolytic and proteolytic activity of trypsin at a concentration of 100 mM. Lineweaver-Burk plots demonstrate that inhibition is competitive, with alpha-aminoacetonitrile being the more potent inhibitor. The enzyme associated with and capable of digesting tropoelastin is inhibited by both lathyrogens when tested against its natural substrate, tropoelastin. Administration of alpha-aminoacetonitrile-HCl to the diet of young chicks (0.1% w/w) resulted in a 62% increase in the yield of tropoelastin and significant reduction in fatality as compared to beta-aminopropionitrile-fumarate.


Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes.

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of N-acetyl-L-glutamate, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well in parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in N-acetyl-L-glutamate concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.


Binding and degradation of insulin by plasma membranes from bovine liver isolated by a large scale preparation.

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes. The insulin binding activity is quite similar to insulin receptors in other cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Nevertheless the Km and the pH dependence of the insulin-degrading activity in both fractions are nearly identical. From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.


Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells.

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10(-6) M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.


N-delta-acetylornithine and S-methylcysteine in blood plasma.

N-delta-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 +/- 0.4 mumol/l (range 0.8--0.2 mumol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 mumol/day). Human blood plasma contains 3.9 +/- 1.9 mumol/l (range 1.4--6.5 mumol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.


In vitro binding of protoheme IX and protoporphyrin IX to components in the matrix of rat liver mitochondria.

The matrix fraction of rat liver mitochondria contains several components capable of binding protoheme IX and protoporphyrin IX in vitro. When binding is analyzed by sucrose density gradient centrifugation, two main groups of components can be separated; i.e. one of high and one of low molecular weight. Experimental evidence is presented indicating a protein nature of these components, and at least six species were detected by gel electrophoresis. The binding of protoheme IX and protoporphyrin IX to matrix components is also evident from a marked change in the heme and porphyrin absorbance spectra in the presence of added matrix. The data obtained indicate that the matrix fraction has a high total binding capacity for heme, but the affinity for heme of the various components seems to be different. Based on the separation of bound and free heme by gel electrophoresis it is concluded that the heme-binding capacity of the high affinity components does not exceed 14 nmol heme . mg-1 protein.


An approximation method for estimating cell loss from a growing tumor.

A simple self-consistent calculational scheme is developed for estimating cell loss for a growing tumor (or other population) when the growth fraction can be estimated at regular intervals. This is applied to published data for a particular much-studied Ehrlich ascites tumor. The loss rate is found to be substantially higher than that estimated by previous, less precise means.


Concanavalin A stimulation of plasminogen activator production in pig kidney cells in culture.

Addition of subtoxic doses of the lectin concanavalin A to growing subconfluent monolayer cultures of pig kidney cells causes an increase in extra- and intracellular plasminogen activator activity which is reversibly inhibited by actinomycin D, cycloheximide and alpha-methyl-D-mannoside. These results suggest that cell surface events may play an important modulatory role in plasminogen activator production.


Bacterial metabolism of propane-1,2-diol.

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO2 by the tricarboxylic acid cycle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.


The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids.

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T4) to T3 and rT3 during iodothyronine secretion might be responsible for the transient fall in the T4/T3 and T4/rT3 ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion. For this purpose T4, T3 and rT3 were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/1 propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T3 and rT3 release while the release of T4 was unaffected. This supports our previous conclusion that T4 is partially monodeiodinated to T3 and rT3 during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T4/T3 and T4/rT3 induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyronine deiodinating processes.


Characterization of mucin isolated from rat tracheal transplants.

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.


Isolation of human pituitary prolactin.

A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenized frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progressive purification was guided by radioimmunoassays. The final product was obtained in yields of 31 microgram/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000-23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophoresis the prolactin preparation was, however, heterogeneous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.


Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria.

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.


[Postluminescence of the marine green alga Ulva fenestrata in millisecond and decisecond attenuation intervals].

Excitation and radiation spectra of the stationary level of postluminescence of marine green alga tallom were measured in millisecond and decisecond attenuation intervals. On the basis of these data and of temperature and light relationships of postluminescence the conclusion is drawn concerning the existence of three independent pigment systems within the range of Photosystem-2, which are responsible for luminescence generation within the given limits of attenuation.


[Radiation entropy and its role in the process of photosynthesis].

The paper deals with the following problems: 1) radiation entropy and the value of maximum performance coefficient of photosynthesizing systems eta m; 2) problem of physical meaning of eta m and on its association with real processes in photosynthesis. It has been shown that for calculating entropy of nonequilibrium radiation its origin should be known and that the value eta m does not practically limit the processes which proceed in photosynthesis.


[Effect of hepatic adhesion factor, contactin, on the mechanical properties of ultrastructural elements of hepatocyte contact].

Injection of isotonic fluid under heightened pressure 20--30 mm of Hg into the bed of the liver reveals inhomogeneity of the mechanical properties of the simple junction of heptacytes: the zone where the divergence of the contact surfaces is observed is interrupted by highly adhesive regions (HAR) where the normal distance between the membranes is preserved. The adhesive factor acts as an organizer of the inhomogeneity of the simple junction: when it is introduced in vivo, new HAR appear in the simple junction. On account of contact-tropic activity of adhesive factor we termed it--contactin.


[Mathematical model of electrogenic transport through biomembranes via oligomeric channels capable of conformational transitions].

A mathematical model of electrogenic ion transport across biomembranes by oligomeric channels liable to conformational transformations has been derived. The model describes changes with time of the membrane potential and near--membrane ion concentrations. Different types of the channel conductance regulation such as activation or inhibition by the permeating ions and membrane potential have been considered. It appears that in the presence of such regulations 1) the channel voltage-current curves have negative resistance regions; 2) the dependence of the quasisteady state (or resting) potential on the ion concentrations in the solution is of hysteresis nature; 3) the model may have multiple steady-state and oscillating solutions.


[Sodium currents in muscles incubated in potassium-free Ringer's solution. Effect of ouabain on unidirectional sodium currents].

Frog sartorius muscles subjected to loading with Na in K-free Ringer solution in the cold were subsequently labelled with 22Na. The uptake of 22Na is not sensitive to ouabain (10(-4) M) while sodium efflux is decreased by oubain. It is concluded that ouabain-sensitive Na-for Na interchange is not present in this condition. Possibly ouabain-sensitive sodium efflux is partly or completely potassium-requiring fraction since some K (approximately 10 microM) is inevitably present in K-free solution. The increase in the rate constant for potassium loss in the presence of ouabain favours this supposition.


[Formation and stability of dissipative structures].

The nonlinear interaction of the disturbed wave packet modes has been analysed within nonlinear parabolic equation. There has also been designed the approach to a limit cycle regime of an isolated mode, the analytical stability criterion of the obtained solutions having separated the existance spheres of isolated and multimode regimes. Stochastization and self-organization processes taking place during the formation of dissipative structures have been studied by methods of random function theory.


[Relationship between incurrent flow, calcium ion concentration and membrane potential].

A dependence of the inward current across the cell membrane giant neurones at garden snail was investigated under voltage champ. It has been concluded that there are two components of the inward current: a calcium-dependent and I0. The latter current probably was carried by sodium ions. The inward Ca current (ICa) is then given as a function [Ca]2+ by: formula (see text) : KCa is a dissociation constant of the sites in outer part channel independent of membrane voltage. The experimental data are interpreted by two barrier membrane model bases of absolute reaction rate Eyring's theory.


[Growth, elastic and inelastic deformations in morphogenetic processes].

On the basis of rheological equation of standard body (as a physical state equation) the stress relaxation or deformation delay (the creep) accompanying the growth of adjacent layers (the model) or the growth of acetabularia apex are studied. It is shown that both initial geometrical characteristics of elastic and viscoelastic deformations determine final morphogenetic complication of bioconstructions, while time characteristics of deformations--the viscosity parameters of growth. Additional morphogenetic switching of development occurs during abrupt (of the phase type) changes in materials states resulting, among other things, from metabolic catastrophs. And vice versa, "catastrophic" changes in the shape of the morphogenetic system affects the course of metabolic processes.


[Temperature transitions of spectrin in solution and in erythrocyte membranes].

Temperature transitions of spectrin in solution and in human erythrocyte membranes are recorded in the region t greater than 40 degrees C by irreversible changes in protein fluorescence spectra. Structural changes are completed 20 min after the sample incubation at an increased temperature. Both for isolated spectrin and for erythrocyte ghosts the temperature of half-transition is 46 +/- 1 degree C. There is no transition in the membranes after the removal of spectrin. Transitions in erythrocyte ghosts and in spectrin solution disappear at pH 5 when spectrin is in an aggregated state. Spectrin is suggested to be responsible for the transitions at 50 degrees C; its state in the cells areas more thermostable than in isolated membranes.


[Chemiluminescent study of the antiradical activity of tocopherol analogs and homologs].

Antioxidative peculiarities of the effect of tocopherol derivatives are considered. Attempts are made to reveal interrelation between tocopherol pharmacological effect and antiradical activity of its derivatives exemplified by an elementary reaction of tocopherol interaction with free peroxide radicals (FR). It is shown that the presence of free hydroxyl groups, number and location of CH3--groups in tocopherol benzol ring produce a significant effect on tocopherol ability to react with FR. The length of lateral phitil chain produces no appreciable effect on the rate of tocopherol reaction with free radicals. The values of energy activation in this reaction are calculated for tocopherol derivatives. Correlation between biological and antiradical activity of tocopherol homologs is shown. The absence of such correlation for tocopherol analogs is explained by the difference in the ability of analogs to be incorporated into biological membranes. Possible tocopherol regulations of the rates of free radical processes proceeding in lipid membranes are considered.


[Regulation of the reaction rate of ATP synthesis in intact mitochondria].

High concentrations of respiration inhibitors are known to sharply decrease the membrane potential in mitochondria. The effect of relatively low concentrations of oxidative phosphorylation inhibitors on the value of membrane potential of intact mitochondria and on the rate of respiration and phosphorylation as well was studied. It has been found that within a certain concentration range the inhibitors of oxidative phsophorylation--malonic acid, sodium cyanide m-chlorophenyl hydrozonecarbonylcyanide, sharply decrease the phosphorylation rate (by 70 divided by 90%) but do not practically a affect the membrane potential value of intact mitochondria in the state 3 according to Chance.


Development of orientation selectivity in the primary visual cortex of normally and dark reared kittens. I. Kinetics.

Kinetics of the development of orientation tuning are inferred from quantitative analysis of extracellular recordings in the primary visual cortex of normally and dark reared kittens. 712 visual cells were classified in three functional groups: a) non-specific cells, and b) immature cells which are not as orientation selective as c) specific cells. Power regression and covariance analysis indicate that the "critical period" begins before 19 days and that the kinetics of the immature pool are the same in both rearing conditions. A catenary process of development of orientation selectivity is proposed, the immature compartment being a transit pool between non-specific and specific cells. Two sequential stages occur: 1) the realisation of an intrinsic programme of maturation, by which cortical specificity appears at eye opening and increases independently of visual experience 2) a phase of "epigenesis" beginning at 19 days, during which functional modification depends on visual experience.


[Heat denaturation of crystalline and dissolved leghemoglobin].

Heat transitions in crystals of leghemoglobin (LH) are studied by means of scanning microcalorimetry and microscopy. It has been found that LH crystals do not melt and their loss of crystal lattice is due to the denaturation of protein globules inside the crystal. Peculiarities of the crystal state (as compared to the solution) are shown in an increase in the cooperative character of heat transition and relaxation time of the system. Subsequent consideration of different variants of correlation of two stages of heat absorption by LH crystals made it possible to determine the type of physical process proceeding in the object by the shape of calorimetric curve. Both observed peaks of heat absorption were grouped with intramolecular processes of different thermodynamic properties. The first peak of heat absorption is a manifestation of intramolecular mobility, both of individual protein segments in relation to each other and of individual segments of alpha-helical regions. Thus microcalorimetry allows a study of peculiar intramolecular dynamics of globular proteins precisely in the crystal state, because the crystal as if synchronizes the movement of individual molecules at the expense of the unification of their kinetic energy, surroundings and mutual orientation.


Development of orientation selectivity in the primary visual cortex of normally and dark reared kittens. II. Models.

The development of orientation selectivity in the primary visual cortex is described by first-order kinetics between three functional compartments chained in a catenary mode. A first model is presented, in which two unidirectional kinetics with constant exchange coefficients, symmetrical in their effects, function in an alternating mode depending on the presence or absence of visual experience. The failure of this model to simulate the modifications induced by a delayed visual experience, when the exchange coefficients are identified to fit normal and dark rearing, supports the hypothesis that the maturation process consequent to interaction with visual environment is dependent on the date at which it is allowed to take place. A second model is then proposed, in which exchange coefficients are piecewise linear functions of time. In order to correctly predict the functional effects of restricted visual experience following prior dark rearing, it is assumed that visuomotor experience during the critical period permits the expression of a non-linear "modifiability gradient" which may have been masked up to this point by the absence of vision or eye movements.


[Synchronous conformational oscillations in the titer of sulfhydryl groups in protein solutions. Reversible oxidation as a possible cause of this phenomenon].

In solutions of creatine kinase, actin and lactic dehydrogenase oscillations of SH-groups are revealed by three independent methods using three different reagents AgNO3, PCMB, DTNB. The variations of the SH-group titre remain after denaturation of the individual protein solution portions. In the solutions of predenaturated proteins no titre oscillations are observed. Possible cause of the observed oscillations of SH-groups titre may be reversible SH--SS transformation.


[Infrared spectroscopic study of DNA--lipid interactions. DNA compacting on disperse particles].

Within the range of relative humidity (r. h.) 0 to 92% there were obtained IR-spectra (4000--900 cm-1) of undeuterated and deuterated films of rat liver lipids, both in the free state and in the complex with native (nDNA) and heat-denaturated DNA (dDNA). Found peculiarities of IR-spectroscopic realization of the complex-formation are explained qualitatively. One of the peculiarities is hyperchromism of lipid bands and hypochromism of the bands of DNA bases. Lipids are shown to despiralize nDNA in the course of their interaction. A suggestion is advanced concerning the nature of basic contacts which form the complex DNA-lipid. It is found that nDNA unlike dDNA produces an inhibiting effect on lipids oxidation and their subsequent hydrolysis. The mechanism of the phenomenon is discussed. The ideas on DNA compactization resulting from its coiling on different protein and nonprotein particles are developed.


[Impulse NMR study of the mobility of water adsorbed onto cellulose].

An impulse method of nuclear magnetic resonance was used for measuring the times of spin-lattice relaxation in the rotating system of coordinates (RSC) for water molecules adsorbed on cottone cellulose. It has been shown that within the temperature region -10 divided by -40 degrees C the spin-lattice relaxation of water in RSC is conditioned by intermolecular interactions modulated with translation movement. The selfdiffusion coefficient of adsorbed water for the sample with 55% humidity at -10 degrees C is determined as 2.0.10(-9) cm2/s and decreases to 0.3.10(-9) cm2s at -40 degrees C, with activation energy of diffusion equalling 8.1 kcal/mol.


Excitation parameters of the repetitive firing mechanism from a statistical evaluation of nerve impulse trains.

Repetitive firing of single tonic neurones is modeled to include in detail both membrane excitation kinetics and electrotonic effects due to membrane non-uniformities in the impulse encoder region. The model is evaluated dynamically and compared with similar data obtained from the crayfish stretch receptor neuron. Two dynamic techniques utilizing small amplitude sinusoidal signals are employed. One technique is used to fix the values of two parameters which relate to the electrotonic control of membrane potential in the interspike interval and to the relaxation time of the K-conductance during repetitive firing. The other technique is employed as a consistency check. The dynamics are particularly sensitive to the K-channel relaxation time in the interspike interval.


[Description of the ratios between metabolite concentrations in a polyenzyme system].

It was demonstrated that the relations between substrate and product concentrations for a reaction catalyzed by michaelian enzyme incorporated in a multienzyme system can be graphically represented by a diverging set of straight lines intersecting in one point, the flux velocity being treated as a parameter. A competitive inhibitor shifts the intersection point along the line of equilibrium state. The relations between the concentrations of more than two reagents are represented by a set of equivelocity surfaces. The relations between substrate and product concentrations for a kinetically cooperative reaction conforming to the graphical representation by the second--order curves were analyzed. The stability criterion was obtained for a multienzyme system with the first enzyme allosterically regulated by products of subsequent reactions.


After hyperpolarization conductance time-course and repetitive firing in a motoneurone model with early inactivation of the slow potassium conductance system.

Early inactivation of the slow potassium conductance system (GK), responsible for the spike afterhyperpolarization (AHP) in spinal alpha motoneurones, has been introduced in a motoneurone model whose GK kinetics give rise to an exponentially decaying AHP conductance. After this modification, the model displays a plateau shaped time-course of the AHP conductance and a faster shortening of the first interval during repetitive firing induced by current steps of increasing intensities. Both features increase the resemblance between the model and the motoneurone behaviour. Comparison with real motoneurones also suggests that GK inactivation may be more developed in "slow" than in "fast" motoneurones.


Study on the transient characteristic in the human visual system using masking experiments.

In this paper the visual masking effect is interpreted on the basic of the transient characteristic in two dimensional neuronal networks. The study investigates the suitability of the effect for use as a measurement method. It is shown that the stimulus distribution in space can be scanned at different points in time and that various dynamic characteristic values of the system can be measured.


The Ornstein-Uhlenbeck process as a model for neuronal activity. I. Mean and variance of the firing time.

Mean and variance of the first passage time through a constant boundary for the Ornstein-Uhlenbeck process are determined by a straight-forward differentiation of the Laplace transform of the first passage time probability density function. The results of some numerical computations are discussed to shed some light on the input-output behavior of a formal neuron whose dynamics is modeled by a diffusion process of Ornstein-Uhlenbeck type.


Neural representation of the acoustic biotope. A comparison of the response of auditory neurons to tonal and natural stimuli in the cat.

Cats were stimulated with tones and with natural sounds selected from the normal acoustic environment of the animal. Neural activity evoked by the natural sounds and tones was recorded in the cochlear nucleus and in the medial geniculate body. The set of biological sounds proved to be effective in influencing neural activity of single cells at both levels in the auditory system. At the level of the cochlear nucleus the response of a neuron evoked by a natural sound stimulus could be understood reasonably well on the basis of the structure of the spectrograms of the natural sounds and the unit's responses to tones. At the level of the medial geniculate body analysis with tones did not provide sufficient information to explain the responses to natural sounds. At this level the use of an ensemble of natural sound stimuli allows the investigation of neural properties, which are not seen by analysis with simple artificial stimuli. Guidelines for the construction of an ensemble of complex natural sound stimuli, based on the ecology and ethology of the animal under investigation are discussed. This stimulus ensemble is defined as the Acoustic Biotope.


Nonlinear dynamics of paleocortex manifested in the olfactory EEG.

The olfactory bulb is the first central component in a highly sensitive yet markedly stable sensory system. It receives a surge of receptor activity with each inspiration and transmits output as a brief burst of oscillatory activity that is most clearly seen in the EEG. These properties together with the known anatomy and physiology of the bulb are used as design criteria to synthesize, evaluate and solve a set of nonlinear differential equations that represent lumped bulbar dynamics. According to the model bulbar processing is in two stages. In the outer layers the interneurons perform the operations of input range compression, integration, clipping, holding, and bias control. In the inner layers the input surge is converted to a burst, which is transmitted by the mitral cells as a pulse density wave. The phase, frequency duration and amplitude of the wave convey information centrally about both the input and the state of the system. The model suffices to replicate the forms of the EEG burst; the pulse probability distributions conditional on the EEG; the waveforms of averaged evoked potentials (AEPs) and post stimulus time (PST) histograms from the bulb and cortex; and the changes in waveform induced by behavioral control of attentiveness and habituation. It is inferred that with selective attention there is a permanent change in the strength of mutually excitatory connections among excitatory neurons, and that with habituation there is a reversible change in the effectiveness of excitatory synapses. The limitations and deficiencies of the model and the need for centrifugal controls of bulbocortical function are discussed.


Expected waiting time for the visual response.

Let the light-quanta be absorbed in the retina according to a Poisson process. The present paper derives bounds for the expected waiting time for an occurrence of a visual response. These bounds can serve as control limits for a proposed coincidence threshold. An upper bound for the probability that a visual response has not occurred in (0,t) is derived and is used to obtain an upper bound for the mean visual threshold.


[Correlation of the increase in DNA methylation and antioxidant activity of mouse liver nuclear lipids after administration of antioxidant and in Ehrlich ascites carcinoma].

The content of 5'-methylcytosine in total DNA of mouse liver increases 2--2,5-fold 3 hrs after a single intraperitoneal injection of antioxidant (4-methyl-2,6-ditretbutylphenol) (20 or 60 mg per 1 kg of body weight) and makes up to 2--2.4 mol.%. The methylation of liver DNA is also increased more than 2-fold in Ehrlich ascite carcinoma. The DNA isolated from mouse liver after administration of antioxidant or during cancer growth markedly differs from liver DNA of intact animals in its CH3-accepting ability under in vitro methylation by the methylase complex from Enterobacter cloacea. The changes in DNA methylation in mouse liver under the effects of antioxidant and in Ehrlich ascite carcinoma are correlated with the changes in the antioxidant activity of liver nuclear lipids.


[Cathepsins D from normal and some human neoplasms].

Cathepsins D were isolated from human liver and spleen, from three malignant tumours (kidney cancer, sarcoma, spleen tumour caused by chronic myeloleucosis) and from one non-malignant tumour (uterine myoma). The isolation procedure involved adsorption on pepstatin-Sepharose and gel-filtration on Sephadex G-100. A comparative study of the properties of these enzymes was carried out. The molecular weights, specific proteolytic activity toward hemoglobin and the synthetic hexapeptide Acetyl-Val-Val-Leu-Ser-Leu-Thr, carbohydrate content and type of dissociation of the molecules into polypeptide fragments during electrophoresis in the presence of DS-Na were determined. All the enzymes under study had molecular weights of about 45 000 except uterine myoma cathepsins (mol. weight 95 000). The latter cathepsin also differed from the other enzymes in its higher carbohydrate content.


[Study of heterogeneity of chromatin endonuclease fragments, using fractionation by step-wise endonucleolysis].

Isolated nuclei from rat liver were incubated at different time intervals under conditions, optimal for manifestation of the Ca, Mg-dependent endonuclease activity. After each of the 6 endonucleolyses chromatin was extracted and 6 chromatin fractions (I--VI) and "residual" chromatin were collected.  A comparative analysis of the "early" (I--III) and "late" chromatin fractions revealed an increased RNA content in the "late" fractions and changes in the composition of the non-histone proteins. Electrophoresis in acrylamide gel concentration gradient demonstrated that fractions I--III predominantly contain high molecular weight fragments whereas fractions IV-VI are represented by highly fragmented chromatin. Each fraction was characterized by peculiar shapes of alkaline denaturation curves and by heterogeneity of charges of their constituent chromatin fragments.


[Lipolytic effects of a synthetic tetradecapeptide, corresponding to 31-44 amino acid sequence of human somatotropin].

A synthetic tetradecapeptide 31--44 developed a fast lipolytic effect in rats and rabbits in vivo and in vitro. The concentration of plasma fatty acid was maximal after 30 min following the injection and came down to the initial level after 60--120 min. The effect was prolonged when the tetradecapeptide was injected in the form of Zn-suspension. Both in rats and rabbits the in vivo lipolytic effect of native somatotropin was pronounced only after a 2-hr latency. Actinomycin D and cycloheximide did not prevent the lipolytic effect of the tetradecapeptide in isolated adipose tissue; however, this effect was not exerted in the absence of Ca2+. The hexapeptide fragment of tetradecapeptide was inactive in rats in vivo, but had a slight effect an isolated rat adipose tissue when taken at high doses. Thus, the lipolytic effect of the tetradecapeptide requires Ca2+ and is more pronounced in rabbits than in rats. Contrary to somatotropin, the effect of tetradecapeptide is fast and independent of RNA and protein synthesis. These data and previously shown tetradecapeptide lipolytic effect on isolated human adipose tissue suggest that the tetradecapeptide possesses structural features required for the lipolytic effect of the whole hormone.


[Reversible precipitation of the glucocorticoid receptor from rat liver by La3+ ions].

It was shown that La3+ ions are capable of precipitating the glucocorticoid receptor from rat liver. Treatment of cytosol containing either free receptor or the hormonereceptor complex with 0,005 M La(NO3)3 results in recptor precipitation. The pellet is readily dissolved in buffer with EDTA. Transcortin and transcortin-like proteins are not affected by La3+ ions. The lanthanium-treated receptor does not lose its ability to bind DNA and chromatin in vitro. It is suggested that precipitation by La3+ ions can be used for separation of the receptor from transcortin and transcortin-like proteins as well as for evaluation of binding parameters for steroid-receptor complexes in rat liver cytosol.


[Use of protein-mediated lipid exchange for studies of membrane-bound enzymes. Properties of monoamine oxidase activated by phosphatidylcholine].

Protein-mediated lipid exchange between phosphatidylcholine liposomes and hepatoma mitochondria was used to alter the lipid composition of the mitochondria and the resulting changes in the activity of monoamine oxidase were studied. Introduction of additional amounts of phosphatidylcholine substantially increased the activity of the enzyme, but did not affect its substrate specificity and the enzyme sensitivity to inhibitors (chlorgyline and deprenyl). The thermal stability of the enzyme was not increased under supplementation of the hepatoma mitochondria with phosphatidylcholine.


[Efflux of Ca2+ from fragmented sarcoplasmic reticulum during AMP deamination].

Deamination of AMP in skeletal muscle sarcoplasmic reticulum followed by an increase in pH from 6,5 up to 8,0 leads in a liberation of part of Ca2+ from the SR vesicles. This effect is enhanced by K+, which activate the deamination, and is suppressed by Mg2+, which inhibit the reaction. The activating effect of AMP on Ca2+ efflux from the vesicles markedly decreases after AMP deaminase dissociation from the vesicles and is restored after reconstitution of their deaminase activity. Substitution of IMP for AMP causes a decrease of Ca2+ efflux from the vesicles. The data obtained are in good agreement with the assumption that the ammonium formation from AMP can favour the release of Ca2+ from some vesicles of SR.


[Two sites of ubisemiquinone binding in mitochondrial succinate oxidase].

The inhibiting effects of several phenol compounds on electron transfer in the respiratory chain of submitochondrial particles were studied. It was shown that the terminal part of the succinate dehydrogenase complex contains a site, which specifically binds the negatively charged phenols (e. g. pentachlorophenol, 2,4-dibromophenol, 2-methoxy-4,6-dibromophenol). The efficiency of the inhibitor anion binding by this site is increased 12-fold after introduction of a methoxy-group into the o-position of 2,4-dibromophenol. Since this site binds both methoxy- and negatively charged phenol groups, it can also act as a possible site of ubisemiquinone interaction with succinate dehydrogenase. Based on the structural similiarity of the b-c1 complex inhibitors, e. g. antimycin, o-hydroxybenzoic acid amides, 2-hydroxy-3-alkyl-1,4-quinones, and ubisemiquinone, an assumption has been made on possible ubisemiquinone binding between cytochromes b and c1. A structural analysis of phenols inhibiting succinate dehydrogenase and the b-c1 complex revealed that in the region between cytochromes b and c1 there act only the inhimitors which contain: a) a negatively charged phenol group; b) a group comprising a heteroatom with an undepleted electron pair and which can act as a ligand, and c) a hydrophobic residue.


[Properties and quaternary structure of ribulosediphosphate carboxylase from the leaves of Elymus (Psathyrosachys) junceus].

The enzyme ribulosdiphosphate carboxylase was isilated from the leaves of Elymus (Psathyrosachys) junceus. The enzyme was found homogenous during disc-electrophoresis in polyacrylamide gel and analytical ultracentrifugation. The sedimentation coefficient for the enzyme is 17,4S. The enzyme molecular weight as determined by the sedimentation equilibrium technique is equal to 540000. The enzyme molecule consists of 2 types of subunits, i.e. the larger subunit has m.w. of 55000, the smaller one--12900. The number of large subunits is 8, that of small ones--8. The specific activity of the homogenous enzyme makes up to 2,45 mkmoles of CO2 per min per mg of protin (pH 8,0, 30 degrees). The purified enzyme was stable in Mg2+- and dithiothreitol-containing buffers for 3--4 weeks at 4 degrees and for 5--6 months at --20 degrees. The amino acid composition of the enzyme molecule is similar to that of the enzyme from spinach leaves.


[Inhibitory effect of verapamil on the acetylcholinesterase activity of skeletal muscle sarcolemma].

It has been found that verapamil reversibly inhibits "in vitro" the activity of membrane--bound and solubilized sarcolemmal acetylcholinesterase. The kinetic analysis has demonstrated a competitive type of inhibition at verapamil concentrations less than 100 mkM and a mixed one at higher verapamil concentrations. The apparent Ki values are similar to or approximately 5,0.10(-5) M and similar to or approximately 3,0.10(-4) M for both types of inhibition, respectively. The effect of vereapamil and Ca2+ on acetylcholinesterase is independent and non-competitive. An increase in the ionic strength leads to a decrease of the verapamil-induced inhibition of acetylcholinesterase. It is suggested that verapamil interacts with the anionic groups of both free and acylated enzyme.


[Molecular structure of aminopeptidase A from bovine kidney].

Using gel-filtration through Sephadex G-100 and polyacrylamide gel electrophoresis in the presence of 0,5% sodium dodecyl sulfate, it was found that aminopeptidase A has a molecular weight of 65 000 +/- 2000 and is made up of two subunits with mol. weights of 33 000 +/- 2000. Each subunit consists of two polypeptide chains with mol. weights of 22 000 +/- 2000 and 12 000. During enzyme dissociation into subunits the aspartylnaphtylamidase activity is lost, while the glutamylnaphtylamidase activity is retained.


[Kinetic patterns of their reactions catalyzed by membrane-bound monoamine oxidase].

The initial rate (v) versus initial substrate concentration [S]0 plots for the reaction of serotonin deamination catalyzed by rat liver mitochondrial monoamine oxidase are characterized by complicated non-hyperbolic patterns. The shapes of the curves in the plots depend on the age and strain of the animals used or on alterations in the physico-chemical state of mitochondria caused by freezing -- thawing and swelling in hypotonic solutions. The v versus [S]0 plots for other substrates (e. g. tyramine) do not obey the Michaelis -- Menten equation as well. In order to interpret the kinetic patterns described a model of the mitochondrial monoamine oxidase structure and function has been proposed. The model is based on the assumption that both catalytic and regulatory allosteric sites are present on the surface of monoamine oxidase molecules. In the process of serotonin or tyramine binding the monoamine oxidases exhibit cooperative properties which are characteristic for the allosteric enzymes. The model also suggests an active regulatory role of the mitochondrial membrane itself.


[Age- and tissue-specific differences in intragenome distribution of 5'-methylcytosine in cow's DNA].

In aged animals the content of 5'-methylcytosine (m5C) in total thymus DNA (1,29 mol.%) and heart muscle (1,10 mol.%) is decreased by 30 and 40%, respectively as compared to that in the 8-months-old embryos. No differences in the GC-content and reassociation kinetics of these DNAs have been revealed. Consequently, the age differences in the m5C content are caused by the decrease in DNA methylation upon ageing. The degree of DNA methylation in various organs (thymus, heart) is different. Upon ageing the degree of genome methylation is decreased and the pattern of methylation is changed: the m5C content is decreased in the repeated sequences and remains practically unchanged in the unique ones. The level of methylation in thymus DNA of moderately (C0t = 4--400) and highly (C0t less than 4) repeated sequences is thereby decreased almost in the same degree. In heart DNA of aged cows the hypomethylation of highly repeated sequences is more pronounced as compared to the moderately repeated ones. The age- and tissue-specific decrease of DNA methylation is regarded as a possible mechanism responsible for the decrease of transcription and functional activity of the cells upon ageing.


[Effect of calcium ions on creatine kinase systems of myocardial cells].

The kinetics of the creatine phosphokinase reaction catalyzed by different creatine phosphokinase (CPK) isoenzymes from the heart and the mechanism of the regulatory action of calcium ions on this reaction have been studied. It has been shown that the kinetic parameters of the reaction in the presence of calcium are similar to those in the presence of magnesium with the exception of the maximal rate of the reverse reaction, which is about three times lower in the presence of calcium. Calcium ions are able to exert a significant regulatory action on the CPK reaction. Simultaneous regulation of the CPK reaction by calcium and magnesium ions can be quantitatively described by a kinetic model, which takes into account the formation of complexes of adenine nucleotides with calcium and magnesium. The mechanism of the CPK reaction regulation by metal ions involves changes in concentrations of the metal -- adenine nucleotide compexes. The regulatory action of calcium on the creatine kinase reaction is the same for all CPK isoenzymes studied.


[Ganglioside composition of normal and leukemic bovine lymphocytes].

The ganglioside composition of bovine peripheral lymphocytes was shown to change sharply under lymphoid leukemia. In normal lymph, lymph nodes, spleen and blood lymphocytes the major ganglioside is N-glycolylhematoside, whereas in calf thymus lymphocytes appreciable amounts of more polar components (GM1- and GD1a-like gangliosides) were found. In leukemic lymphocytes isolated from the same tissues the hematoside content is decreased, while the amount of more polar gangliosides is increased. Possible causes of the altered ganglioside pattern in leukemic lymphocytes are discussed.


[Induction of the microsomal monooxygenase system of rat liver by combined administration of testosterone and phenobarbital].

A comparative study of the ability of phenobarbital, testosterone and their combination to induce the liver microsomal monooxygenase system after 9-day administration of these compounds to intact male and female rats was carried out. It was shown that administration of testosterone does not increase the level of cytochromes P450 and b5 in the livers of male and female rats. However, after a combined administration of the two compounds testosterone significantly enhances the inducing effects of phenobarbital (i. e. superinduction) in female rats; no such effect was observed in the livers of male rats. The rates of oxidation of hexobarbital, ethylmorphine and testosterone by liver microsomes are also increased after a combined administration of the two inducers. However, the additive effects of the two substances on substrate oxidation are observed when the latter was calculated per mole of cytochrome P450. An administration of testosterone to male rats does not result in an increase of the rate of hexobarbital and testosterone oxidation by isolated liver microsomes.


[Membrane proteins of chloroplasts of intact and TMV-infected tobacco plants].

The effects of viral infection on the membrane proteins from tobacco plant chloroplasts differing in their stability for TMV infection were studied. It was shown that the changes in the chloroplasts of labile and resistant tobacco varieties are oppositely directed. The data from amino-acid analysis, SH-group determination and infrared spectra of the membrane proteins of the resistant variety are indicative of conformational changes caused by disruption of the hydrogen bonds, which stabilize the protein, by changes in aggregability, etc. due to infection and metabolic disturbances in the infected cell. The conformational changes in the chloroplasts of the stable variety are adaptive and affect the biological activity, enzymatic and immunological properties and energy metabolism of the chloroplasts.


[Filtration-fluorescence determination of DNA sedimentation profiles].

A method for determination of DNA sedimentation profiles in density gradients of sucrose or salts is proposed. The method consists in isolation and purification of DNA from the fractions by molecular filtration and a subsequent determination of DNA content by the fluorescence of the DNA-ethidium bromide complex.


[Purification and some physico-chemical properties of myocardial adenylate deaminase].

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.