bluuebunny/crossref_metadata_2025 huggingface dataset:
In cultured chick embryo fibroblasts the hexose transport components are not the 75 000 and 95 000 dalton polypeptides synthesized following glucose deprivation
Authors: Zala Cedric A. , Salas-Prato Milagros , Yan Wing-Tat , Banjo Batya , Perdue James F.
Published: December 2009

Glucose deprivation of chick embryo fibroblasts results in a cycloheximide-sensitive stimulation of hexose transport and an increase in the levels of glucose-regulated polypeptides of molecular weights 75 000 and 95 000. The relationship between these two phenomena is evaluated in this study. The glucose deprivation-induced stimulation of hexose transport was observed to occur in two phases: a rapid (complete by 15 min) cycloheximide-insensitive increase of 50–100% and a slower (observable by 6 h) cycloheximide-sensitive increase in transport to about five times the basal level. The time course of the latter increase preceded that of the appearance of the 75 000 and 95 000 dalton polypeptides; by the time that increases in the levels of these polypeptides were observed, the hexose uptake rates had almost reached their maximum value. Upon cellular fractionation, the greatest enrichment of the 75 000 and 95 000 dalton polypeptides was observed in the endoplasmic reticulum fraction, which was devoid of vesicular stereo specific D-glucose uptake activity. The plasma membrane fraction was enriched in stereospecific D-glucose uptake activity but not in the 75 000 and 95 000 dalton polypeptides. The glucose deprivation-induced increase in hexose uptake was not prevented by tunicamycin, although this inhibitor of protein glycosylation decreased the hexose uptake of glucose-fed cells by 80% after 24 h. However, under these latter conditions an increase in the levels of the 75 000 and 95 000 dalton polypeptides was observed. On the basis of this data, we conclude that the polypeptides of molecular weights 75 000 and 95 000 are not involved in glucose transport.
Activation of ouabain-sensitive p-nitrophenylphosphatase by carbachol and cGMP in rat submandibular gland
Authors: Shi Maggie M. , Stewart D. J. , Sen A. K.
Published: December 2009

Na+,K+-ATPase activity was monitored by measuring ouabain-sensitive K+-dependent p-nitrophenylphosphatase (p-NPPase) activity in rat submandibular gland slices. Carbachol (carbamylcholine chloride) stimulated the p-NPPase activity in the presence of calcium but not in its absence. Carbachol activation of the enzyme was totally ouabain sensitive and could be blocked by atropine. A minimal requirement of sodium ion extracellularly was required for this carbachol stimulation. cGMP and its dibutyryl analogue was also effective in stimulating the enzyme activity, whereas, cAMP was ineffective. Calcium, however, was not required for cGMP activation of the p-NPPase activity. The result indicates that calcium is the second messenger and cGMP is the tertiary connection between cholinergic stimulation and Na+,K+-ATPase activation in these glands. Activation of Na+,K+-ATPase is postulated to be responsible for primary fluid formation.
Enrichment of sulfogalactosylalkylacylglycerol in a plasma membrane fraction from adult rat testis
Authors: Shirley Margaret A. , Schachter Harry
Published: December 2009

Adult rat testis homogenates were fractionated by differential centrifugation followed by two discontinuous gradient centrifugation steps under identical conditions except for the absence of digitonin in the first gradient and the presence of 0.03% digitonin in the second gradient. The first gradient centrifugation yielded a membrane fraction enriched 28.8-fold in 5′-nucleotidase, 21.5-fold in UDP-Gal:GlcNAc galactosyltransferase and 18.6-fold in UDP-GlcNAc:α-D-mannoside N-acetylglucosaminyltransferase. Repeat centrifugation of this membrane fraction in the presence of digitonin resulted in the sedimentation of most of the membrane material to a denser level of the gradient; this material was enriched 32.1-fold in 5′-nucleotidase but only 1.9-fold in galactosyltransferase and 8.4-fold in N-acetylglucosaminyltransferase. The plasma membrane fraction was shown to be free of glucose-6-phosphatase, succinate dehydrogenase, β-N-acetylglucosaminidase, DNA, and RNA. The fraction therefore appears to be enriched in plasma membrane but relatively free of Golgi membrane contamination, as indicated by the relatively low levels of glycosyltransferases, and of contamination by other organelles. The testicular cells which contribute plasma membrane to this fraction have not yet been definitively identified; the contribution by Sertoli cells is particularly difficult to assess since these cells have been reported to be enriched in 5′-nucleotidase. However, sulfogalactosylalkylacylglycerol (SGG), a lipid previously shown to be present primarily in primary spermatocytes, spermatids, and spermatozoa, was enriched 33.1-fold in the plasma membrane fraction; this finding as well as experiments with [36S]sulfate-labeled sulfogalactosylalkylacylglycerol at various times after injection of radioactive label have indicated that both spermatocytes and spermatids were contributing SGG-rich membrane material to our plasma membrane preparation. This membrane material is most probably derived from the plasma membranes of the spermatocytes and spermatids.
Procion dyes as affinity ligands and reporter groups for dihydrofolate reductase from Walker 256 carcinoma
Authors: Johnson S. J. , Stevenson K. J. , Gupta V. S.
Published: December 2009

Procion dye – agarose matrices were investigated for isolation of dihydrofolate reductase (FAH2R) from Walker 256 carcinosarcoma. Cibacron blue F3GA, Procion blue MX4GD, Procion blue HERD, and Procion red H3BN covalently bound to agarose adsorbed greater than 85% of pure FAH2R from 100 mM imidazole buffer, pH 6.3, and this enzyme was specifically and quantitatively eluted with 1 mM folate. The capacity and selectivity of the dye–agarose matrices were greater at low dye incorporation. Difference spectroscopy of the FAH2R – Cibacron blue F3GA complex indicated that 2 mol of the dye were bound in hydrophobic environments with each mole of the enzyme. NADPH and folate (at twofold molar excess over enzyme) or 1 M KCl displaced only 1 mol of Cibacron blue F3GA. This dye interacted stoichiometrically in a specific manner with the active site of FAH2R probably spanning the folate and NADP binding sites. The second dye molecule appears to be bound in a nonspecific hydrophobic manner. Selected Procion dye – agarose matrices can be used for partial purification of FAH2R from tumor homogenate.
Cholesterol metabolism in obese mice
Authors: Hahn Peter
Published: December 2009

Young male obese mice and their lean litter mates (strain ob/ob) were compared. Serum cholesterol levels were higher in obese than in lean animals. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was lower in brown adipose tissue and liver of obese mice than in tissue from their lean litter mates. However, activity of this enzyme was found to be the same or higher in white adipose tissue of obese animals than in their lean litter mates. During complete starvation, blood cholesterol levels in obese mice decreased and attained the lower level of lean ones after 48 h. After 24 h of starvation, enzyme activity decreased in white fat of obese mice only. Simple calculations indicate that white fat from obese mice produces about fourfold more cholesterol per day per unit body weight than does the same tissue from lean mice.
In vitro studies on L-serine deaminase activity of Escherichia coli K12
Authors: Newman E. B. , Kapoor V.
Published: December 2009

Extracts of Escherichia coli K12 contain an enzyme which deaminates L-serine. This serine deaminase appears to be a soluble enzyme and is inhibited by substrate analogues, metal ions, and chelators. The activity, which is very unstable in vitro, is protected, and in some cases, even activated by substrate, substrate analogues, and by ferrous ion. The enzyme has proved unstable in all attempts at purification. It resembles closely the L-serine deaminase activity in other microorganisms, but is very different from the mammalian enzyme. As judged by comparison with organisms in which this enzyme serves as part of the principal carbon-handling pathway, L-serine deaminase activity is present in E. coli extracts in physiologically significant amounts.
The chemical modification of Escherichia coli ribosomes with methyl p-nitrobenzenesulfonate. Evidence for the involvement of a histidine residue in the functioning of the ribosomal peptidyl transferase
Authors: Glick Bernard R.
Published: December 2009

Free 70S ribosomes and 70S ribosome – AUG – f Met-tRNA complexes were reacted, separately with methyl p-nitrobenzenesulfonate. This reagent inhibits the peptidyl transferase activity of 70S ribosome – AUG – fMet-tRNA but not of free 70S ribosomes. The data suggest that methyl p-nitrobenzenesulfonate reacts with a unique site which is a part of the ribosomal peptidyl transferase.
Protection of actin against proteolysis by complex formation with deoxyribonuclease I
Authors: Burtnick L. D. , Chan K. W.
Published: December 2009

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-67 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.
Relationship between chromatin structure and replication in mouse L-cells
Authors: Sheinin Rose , Setterfield G. , Dardick I. , Kiss G. , Dubsky M.
Published: December 2009

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.
The uptake and acylation of exogenous lysophosphatidylethanolamine by Escherichia coli cells
Authors: Hellion P. , Landry F. , Subbaiah P. V. , Proulx P.
Published: December 2009

Escherichia coli envelopes were fractionated to yield inner and outer membrane fractions. Both these fractions were found to convert [14C]lysophosphatidylethanolamine to its diacyl analogue. Intact Escherichia coli cells were capable of absorbing exogenous labelled lysophosphatidylethanolamine and converting it to phosphatidylethanolamine. When the 14C- and 32P-labelled lyso analogue was used, both the absorption process and the conversion to diacyl analogue proceeded without a significant change in isotope ratio either in the presence or in the absence of added inorganic phosphate. The absorption process was not markedly stimulated by Ca2+ in the medium; it proceeded to an amount representing 25–30% of the endogenous membrane lipid and was accompanied by some degradation to water-soluble products which accumulated in the cell mainly, but also in the incubation medium. The absorbed lipid was recovered in both the inner and outer membrane fractions of the cell envelope. The results indicate that Escherichia coli inner and outer membranes are capable of absorbing exogenous lysophosphoglyceride and converting it into structurally useful diacyl analogue.
Plasma membrane mediated thymidine transport in AKR spleen cells
Authors: Strauss Phyllis R. , Sheehan James M. , Taylor Judith
Published: December 2009

In this paper we characterize the thymidine transport systems in nonadherent spleen cells from normal leukemic (AKR) mice and from AKR mice which have been stimulated in vivo with concanavalin A (Con A). We have shown that splenic lymphocytes from normal AKR mice transport thymidine (two kinetic components, Km values of 34 μM and 1.6 mM) whereas lymphoid cells from C57L/J and outbred (CD-1) mice do not. Following Con A stimulation of AKR mice, three components (Km values of 6 μM, 212 μM, and millimolar range) were observed. The current data should be compared with previously published results for splenocytes from Con A stimulated CD-1 mice. Although those cells transport thymidine with two kinetic components (Km values of 160 μM and 4 mM), they lacked the lowest Km system present in AKR splenocytes.Thymidine transport was also examined in lymphocytes from several AK × L recombinant inbred mouse strains derived from the cross AKR/J × C57L/J. Two strains which lacked MuLV did not show time-dependent thymidine translocation whereas two strains which possessed MuLV demonstrated time-dependent thymidine translocation. Moreover, cells from the congenic strain L.AKR-Akv-2, which carried the Akv-2 genome on a C57L background, also showed thymidine transport. Thus a unique ability to transport thymidine can be correlated with the presence of the murine leukemia virus genome.
Effects of phenobarbital upon triacylglycerol metabolism in the guinea pig
Authors: Goldberg David M. , Yu Alexander , Roomi M. Waheed , Roncari Daniel A. K.
Published: December 2009

The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male guinea pigs injected intraperitoneally with 50 mg phenobarbital∙kg−1 for 7 days. Enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity, cytochrome P450 content, and hepatic γ-glutamyltransferase activity. Increased activity of γ-glutamyltransferase was also observed in the blood serum of treated animals. The phenobarbital-treated guinea pigs manifested increased hepatic triacylglycerol content and serum triacylglycerol concentration, accompanied by enhanced ability of cell-free fractions of liver to synthesize glycerolipids in vitro from sn-[14C]glycerol 3-phosphate and fatty acids. Microsomal phosphatidate phosphohydrolase accounted for 97% of the total liver activity of this enzyme, and its specific activity was 50-fold higher than that of the cytosolic enzyme when each was measured under optimal conditions. Activity of the cytosolic phosphohydrolase per liver doubled and that of the microsomal phosphohydrolase increased by 40% in the phenobarbital-treated guinea pigs. The microsomal, but not the cytosolic enzyme, showed a significant correlation with hepatic triacylglycerol content. Significant correlation was observed between the various parameters of hepatic microsomal enzyme induction and hepatic triacylglycerol content, suggesting that enzyme induction may promote triacylglycerol synthesis and consequent hypertriglyceridaemia in the guinea pig.
Regulation of the F1-ATPase from mitochondria of Vigna sinensis (L.) Savi cv. Pitiuba by spermine, spermidine, putrescine, Mg2+, Na+, and K+
Authors: Peter Horst W. , Pinheiro Moema Rodrigues , Lima Maria Silva
Published: December 2009

Mitochondria from Vigna sinensis (L.) Savi cv. Pitiuba contain the polyamines spermine, spermidine, and putrescine. The membrane-bound F1-ATPase from mitochondria of Vigna sinensis is activated by these polyamines at physiological concentrations. The effect of polyamines on the membrane-bound F1-ATPase is dependent on the concentrations of Na+, K+, MgATP, and Mg2+. Excess Na+ or K+ prevents the activation of the membrane-bound F1-ATPase by spermine and spermidine, but not by putrescine. The most pronounced effects were observed at low MgATP concentrations in the absence of Na+ and K+. At [MgATP] = 0.08 mM, spermine activation of the membrane-bound F1-ATPase was 130%.The membrane-bound F1-ATPase is slightly activated by Mg2+ at lower concentrations and strongly inhibited by Mg2+ at higher concentrations. Activation as well as inhibition is dependent on the substrate MgATP concentration. Although there is competition between Mg2+ and MgATP, the binding sites for these two ligands are different (pseudocompetitive inhibition). The inhibition of the membrane-bound F1-ATPase can be reversed by polyamines. There is evidence that the binding sites for Mg2+ and polyamines are identical.The F1-ATPase detached from the membrane is neither activated by polyamines nor inhibited by Mg2+. Therefore, the binding sites for Mg2+ and polyamines seem to be localized on the membrane.
Biosynthesis and characterization of heat shock proteins in Chironomus tentans salivary glands
Authors: Tanguay R. M. , Vincent M.
Published: December 2009

The protein synthesis response of Chironomus tentans salivary glands to a heat shock was analyzed by means of [3H]leucine pulse labeling, sodium dodecyl sulfate – polyacrylamide slab gels, and fluorography. Seven new cytoplasmic polypeptides with relative masses (Mr) of 90 000), 76 000, 73 000, 68 000, 28 000, 25 000, and 22 000 are synthesized in response to a39 °C heat shock (HS) while the synthesis of most of the normal proteins is reduced. The major HS-induced protein is the 68 000 species. The length of the heat treatment does not modify markedly the pattern of induced proteins but the temperature has an effect: while the responses at 37 and 39 °C are similar, two HS-induced polypeptides (Mr 90 000 and 76 000) are not seen after a treatment at 41 °C. Kinetic studies of the response show an asynchrony in the appearance of the various HS proteins indicating that their individual rates of synthesis differ or that their induction is not fully coordinated. No significant differences were found between the protein patterns of a mitochondria-rich cytoplasmic zone obtained by microdissection and the whole cytoplasm, but two heat-induced proteins are present in the microdissected nuclei: a 68 000 polypeptide, comigrating with the major cytoplasmic one, and a 34 000 protein, almost exclusively seen in the nucleus.
Energy coupling mechanisms in host-grown Mycobacterium lepraemurium
Authors: Ishaque M. , Adapoe C. , Kato L.
Published: December 2009

Energy coupling mechanisms of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata were investigated. Cell-free extracts catalyzed phosphorylation coupled to the oxidation of generated NADH, added NADH, and succinate yielding P/O ratios of approximately 0.8, 0.6, and 0.4, respectively. Ascorbate oxidation alone or in the presence of cytochrome c or N,N,N′,N′-tetramethyl-p-phenylenediamine was not coupled to ATP synthesis.The oxidative phosphorylation was completely uncoupled by 2,4-dinitrophenol, 2,6-dibromophenol, pentachlorophenol, m-chlorocarbonylcyanide phenylhydrazone, dicumarol, and gramicidin at concentrations which did not cause any inhibition of oxygen uptake. While the NADH oxidation and associated phosphate esterification was markedly sensitive to rotenone and other flavoprotein inhibitors, these inhibitors had no effect, however, on the phosphorylation coupled to succinate oxidation. The respiratory chain inhibitors such as antimycin A or 2-n-heptyl-4-hydroxyquinoline-N-oxide, and cyanide were the potent inhibitors of the phosphorylation associated with the oxidation of NADH and succinate. The ATP formation coupled to the oxidation of NADH and succinate was also inhibited by oligomycin as well as by the thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide. The results indicated that NADH and succinate oxidation by in vivo grown M. lepraemurium was mediated by oxidative enzymes involving first and second energy coupling sites.
The anomeric specificity of β-galactosidase and lac permease from Escherichia coli
Authors: Huber R. E. , Hurlburt K. L. , Turner C. L.
Published: December 2009

β-Galactosidase was found to act on α-lactose slightly more than twice as rapidly as on β-lactose for both the hydrolysis and transgalactosylis reactions. The effect was shown to be on the Vmax values; the Km values for the different anomeric forms were the same. The step of the reaction for which the enzyme has anomeric specificity was shown to be glycosidic bond breakage. The steps in glucose release or in the glucose acceptor reaction were not affected by anomeric composition. Neither allolactose hydrolysis nor transport of lactose into the cells by lac permease was sensitive to the anomeric composition of the substrate. The implications of these results for lac operon induction and for lactose metabolism are discussed.
The mechanism of replication of XVII. Purification and partial characterization of the gene A and A* proteins
Authors: Dubeau Louis , Hours Christian , Denhardt David T.
Published: December 2009

A simple method for purifying the gene A plus A* proteins of bacteriophage [Formula: see text] is presented. The proteins are relatively hydrophobic and precipitate from aqueous solutions at low ionic strength. The gene A plus A* protein preparation was able to stimulate [Formula: see text] DNA replication in vitro and was able to nick supercoiled [Formula: see text] replicative form I to produce unit-length linear and circular strands of DNA. The kinetics of the nicking reaction were more consistent with a stoichiometric than with a catalytic mechanism. Under nondenaturing conditions the presumably globular A and A* proteins sedimented with apparent molecular weights of 110 000 and 90 000.
Similar sets of terminal oligonucleotides from reovirus double-stranded RNA and viral messenger RNA synthesized in vitro
Authors: Hastings Kenneth E. M. , Millward Stewart
Published: December 2009

Reovirus genomic double-stranded RNA (dsRNA) and viral messenger RNA synthesized in vitro were labeled by periodate oxidation and [3H]borohydride reduction. The 3H label was incorporated into 3′-terminal C residues and 5′-terminal N7-methylguanosine residues. Analysis of ribonuclease digests of the 3H-labeled RNA indicated that the minus strands of dsRNA contained the common 3′-terminal sequence [Formula: see text]PypPupGpC and that the plus strands contained heterogeneous sequences at both the 5′ termini and 3′ termini that corresponded to sequences at the 5′ and 3′ termini of mRNA. These results suggest that the plus strands of dsRNA and mRNA synthesized in vitro are essentially identical sets of molecules.
Cloning and expression of the fumarate reductase gene of Escherichia coli
Authors: Lohmeier Elke , Hagen D. Scott , Dickie Peter , Weiner Joel H.
Published: December 2009

Mutants of Escherichia coli deficient in fumarate reductase activity and therefore unable to grow anaerobically with fumarate as an electron acceptor have been isolated. By F+-mediated conjugation and complementation with the mutant host, two E. coli: Col E1 recombinant DNA plasmids have been identified from the Clarke and Carbon Colony Bank which carry the structural genes for fumarate reductase. Bacteria harboring either of these plasmids express about ten times the normal level of fumarate reductase. Enzyme purified from the two sources, plasmid-carrying and plasmidless E. coli, have identical physical and kinetic properties indicating that both the 69 000 and 25 000 dalton polypeptides are amplified. Regulation of plasmid-encoded enzyme, like the chromosomally encoded enzyme, is dependent upon the presence of fumarate and anaerobiosis.
The association of intercellular structural glycoproteins with lipids in canine cartilage
Authors: Chopra Ravi K. , Bowness J. Michael
Published: December 2009

Two noncollagenous insoluble structural glycoprotein fractions (A and G), from uncalcified canine puppy rib cartilage, each contained about 8% of extractable lipid. This percentage of lipid is higher than that found in any of the other tissue fractions obtained by the preparative procedure used. The composition of the lipid extracted from the structural glycoproteins is quite distinct from the lipid in the guanidine∙HCl extract of cartilage, the lipid of cartilage matrix vesicles, and that of other known lipid membranes. On ultracentrifugation, solubilized A or G, obtained by using 50 mM dithiothreitol (DTT) in 5 M guanidine∙HCl followed by dialysis, showed a floating fraction (density 1.14–1.16 g/mL) which was decreased by prior delipidation and increased by lipidation. Chromatography of [3H]palmitate, [14C]cholesterol, or [14C]phosphatidylcholine on Sepharose 2B showed that their elution behaviour is altered in the presence of solubilized A or G. All the labelled lipids cochromatograph, but only with A or G protein in the high molecular weight region. After boiling the low density ultracentrifugation fraction with 1% sodium dodecyl sulfate (SDS), 8 M urea, and 50 mM DTT, disc gel electrophoresis showed that it contained the same subunits as the high density fraction. It is concluded that lipids can readily form a complex with a specific fraction of A or G. This fraction may be a combination of several undissociated subunits or an undenatured form of the proteins. The complex is stable enough to be of possible significance in the metabolism of connective tissues. No antigenic relationships have been found between the cartilage structural glycoproteins or cartilage extracts and the plasma lipoproteins LDL, VLDL, and Lp(a).
Lipid composition of lung and lung lavage fluid from map turtles (Malaclemys geographica) maintained at different environmental temperatures
Authors: Lau Ming-Jarm , Keough Kevin M. W.
Published: December 2009

The phospholipids of lung and lung lavage fluid from map turtles (Malaclemys geographica) acclimated to 5, 14, 22, and 32 °C have been analyzed. The yield of surfactant (lavage) lipid P was higher at 32 than at 5 °C, but there was no significant change in lung lipid P. The relative proportions of phospholipids from the lung were PC > PE > SM > PS > PI, while those for the lavage were [Formula: see text]. PC constituted 71–78% of the surfactant phospholipid over the temperature range. Small changes in the amounts of PC and PE of lung and in PC and PI of lavage were observed at different temperatures. The fatty acid compositions of the lipids from the two sources were different in many respects. The lavage lipids contained more saturated acids, especially palmitic acid, than did the lung lipids. The fatty acid compositions indicated the presence of disaturated PC in lung lavage and diunsaturated species of PC and PE in lung. The total saturated fatty acids of PC and PE in lung and of PC in surfactant increased at higher temperatures. In the surfactant PC this increase reflected principally a rise in palmitate and a drop in palmitoleate contents. The saturated PC obtained from lavage at each temperature contained more than 85% palmitic acid, indicating that the principal saturated PC was DPPC. These changes are consistent with the type of alterations in the lipid compositions of membranes which occur during homeoviscous adaptation.
Partial purification and characterization of the components of the neutral ribonucleage II – inhibitor system of normal and dystrophic mouse skeletal muscle
Authors: Little Brian W. , Meyer William L.
Published: December 2009

The complexes between a proteinaceous inhibitor and neutral ribonuclease II (EC 3.1.27.5) purified from low ionic strength extracts of normal and dystrophic mouse muscle are essentially indistinguishable in (a) purification behavior, (b) apparent molecular weights of approximately 50 000, (c) thermal denaturation (50% loss of activity in 5 min at 73.5 °C), (d) isoelectric points (pH 4.8), and (e) procedures for reversible resolution into free inhibitor and free RNase II.The free RNase II species are also similar whether obtained by resolution of the purified complexes or by direct isolation of free enzyme from dystrophic muscle. All have apparent molecular weights of 11 500 compared with 13 700 for bovine pancreatic RNase A; all retain 80% of activity after 5 min at 95 °C. The active RNases II prepared directly from muscle, by resolution of inhibitor complexes or by organic mercurial treatment of the inhibitor complexes, all have identical pH–activity profiles in 200 mM KCl with an optimum near pH 7.0. In comparison RNase A has an optimum pH near 7.5 and its activity decreases more rapidly as KCl concentration is increased above 50 mM KCl.RNase II inhibitor obtained by resolution of the purified complexes or by direct isolation in the free form from normal muscle extracts has an apparent molecular weight of 42 000 and is very sensitive to heat; it loses all activity at 40 °C in 5 min.These studies (a) provide methods for obtaining useful amounts of the components of the neutral RNase II – inhibitor system from muscle, (b) provide the first method reported for the reversible resolution of RNase II – inhibitor complexes, (c) fail to show any distinct difference between corresponding components of the system from normal and dystrophic mice, (d) establish interesting differences between the apparently homologous enzymes, murine muscle neutral RNase II, and bovine pancreatic RNase A, and (e) provide a substantially lower molecular weight estimate for RNase II inhibitor from muscle than has been reported for the inhibitor from liver, kidney, and placenta.
The similarity of histones from turtle erythrocytes and liver
Authors: Rutledge R. G. , Shay C. E. , Brown G. L. , Neelin J. M.
Published: December 2009

Using the fresh-water "red-eared turtle" Pseudomys scriptans elegans, we have confirmed the existence of a minor component H1s among the lysine-rich histones of turtle erythrocytes by three forms of gel electrophoresis and two forms of chromatography. It was separated from the major components by both cation-exchange chromatography and molecular-exclusion chromatography and shown to differ slightly but significantly in content of several amino acids as well as being shorter than the other lysine-rich histones. Although its composition is close to that of the "tissue-specific" F1b of sea turtle erythrocytes, it is present in even greater relative amount in red-eared turtle livers. In most respects it resembles the satellite histone H10 of mammalian tissues. No unusual histones were observed among the perchloric acid insoluble histones of turtle erythrocytes.
Comparison of the structure and function of ribulosebisphosphate carboxylase–oxygenase from a cold-hardy and nonhardy potato species
Authors: Huner Norman P. A. , Palta Jiwan P. , Li Paul H. , Carter John V.
Published: December 2009

A comparison of ribulosebisphosphate carboxylase–oxygenase from the leaves of the non-acclimated, cold-hardy species, Solanum commersonii, and the nonacclimated, nonhardy species, Solanum tuberosum showed that this enzyme from the two species differed in structure and function. The results of sulfhydryl group titration with 5,5′-dithiobis(2-nitrobenzoic acid) indicated that the kinetics of titration and the number of accessible sulfhydryl groups in the native enzymes were different. After 30 min, the enzyme from the hardy species had 1.7 times fewer sulfhydryl groups titrated than that from the nonhardy species. In the presence of 1% (w/v) sodium dodecyl sulfate, the total number of sulfhydryl groups titratable with 5,5′-dithiobis-(2-nitrobenzoic acid) was the same for both species. However, this denaturant had a differential effect on the kinetics of titration with 5,5′-dithiobis(2-nitrobenzoic acid). Both enzymes had a native molecular weight of about 550 000. The quaternary structures of the two enzymes were similar with the presence of large and small subunits of 54 000 and 14 000, respectively. However, there was more polypeptide of 108 000 – 110 000 present in preparations of the enzyme from S. tuberosum than from S. commersonii. This polypeptide is an apparent dimer of the large subunit on a relative mass basis. The large subunit of the enzyme from S. tuberosum was more sensitive to the absence of reducing agent and was more sensitive to freezing and thawing than the large subunit of the enzyme from S. commersonii. Catalytic properties of both enzymes at 5 and 25 °C indicated no significant difference in the [Formula: see text] at either temperature. However, the Vmax at 5 °C for the enzyme from S. commersonii was 35% higher than that of the enzyme from S. tuberosum. In contrast, the Vmax at 25 °C for the enzyme of the hardy species was 250% lower than that of the enzyme from the nonhardy species.
Site of synthesis of metallothionein in rat liver
Authors: Cherian M. George , Yu Sharon , Redman Colvin M.
Published: December 2009

Free and membrane-attached polysomes were isolated from the livers of normal and cadmium-treated rats, and were translated using L-[35S]cysteine and a nuclease-treated reticulocyte lysate system. The translation products were analyzed for radioactive metallothionein by immunoprecipitation with antibodies to rat cadmium metallothionein followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. In both normal and cadmium-treated rats, radioactive metallothionein was produced by free polysomes but not by membrane-attached polysomes. Cadmium treatment did not increase the in vitro ability of polysomes to synthesize metallothionein. As a control, the translation products of these two classes of polysomes were also analyzed for radioactive albumin and it was confirmed that membrane-attached polysomes produce albumin but do not synthesize metallothionein. The cell-free synthesis of metallothionein by free polysomes was also demonstrated by isolation of nascent metallothionein by Sephadex gel filtration and DEAE-cellulose chromatography. In adult rat liver there are two forms of metallothionein and both were produced in vitro by free polysomes.
Effect of glutathione deficiency on the pool of CoA–glutathione mixed disulfide in Escherichia coli
Authors: Loewen Peter C.
Published: December 2009

The formic acid extracts of several glutathione-deficient strains of Escherichia coli have been assayed for the presence of the mixed disulfide of CoA and glutathione, CoASSG. Strains deficient in γ-glutamyl-cysteine synthase (EC 6.3.2.2) produced only CoA dimer. Strains deficient in glutathione synthase (EC 6.3.2.3) produced the mixed disulfide of CoA and the γ-glutamyl-cysteine dipeptide. The pool size of total CoA in the cell did not change significantly even in the absence of glutathione.
Presence of γ-glutamyltransferase in the renal microvascular compartment
Authors: Dass P. D. , Misra R. P. , Welbourne T. C.
Published: December 2009

The association between the brush border enzyme alkaline phosphatase and γ-glutamyltransferase was determined by sucrose density gradient analysis of crude kidney homogenates, isolated glomeruli, and isolated microvessels. As previously established there is an overlap of these enzyme activities in the crude homogenate corresponding to a density of 1.17 g∙cm−3. In contrast, isolated glomeruli sedimented with a peak of 1.25 g∙cm−3 and exhibited γ-glutamyltransferase activity but little alkaline phosphatase activity; homogenizing isolated glomeruli shifted the fragments to a density coincident with that observed for the crude homogenate γ-glutamyltransferase peak. A second population of capillaries, isolated microvessels, were homogenized and analyzed on the sucrose density gradient. These fragments sedimented over the same range as crude homogenate γ-glutamyltransferase peak but were devoid of alkaline phosphatase activity and yet exhibited remarkable γ-glutamyltransferase activity. The results indicate homogenization of renal cortex results in a heterogenous collection of particles from both tubular and microvascular locations exhibiting γ-glutamyltransferase activity which overlap with the brush border alkaline phosphatase containing membranes. However, isolation of microvessels and glomeruli prior to homogenization allows separation of γ-glutamyltransferase from alkaline phosphatase activity; between 10 and 20% of the total homogenate γ-glutamyltransferase activity is estimated to be associated with the microvascular compartment.
Regulation of the dephosphorylation of glycogen phosphorylase a and synthase b by glucose and caffeine in isolated hepatocytes
Authors: Kasvinsky Peter J. , Fletterick Robert J. , Madsen Neil B.
Published: December 2009

Synergistic regulation of glycogen phosphorylase a by the competitive inhibitors glucose and caffeine in vitro indicates a possible physiological role for the negative effector site which binds caffeine (nucleoside site). In intact viable hepatocytes glucose promotes the phosphorylase a to b conversion by phosphorylase phosphatase. This conversion is considered to be a necessary prelude to the activation of glycogen synthase by phosphatase and of importance in hepatic regulation of glucose homeostasis. The effects of glucose and(or) caffeine on the conversion of phosphorylase a to b and synthase b to a were studied. Assays of phosphorylase a were used which limited synergistic inhibition (in the assay) by these ligands. Such an approach is necessary to achieve an accurate measure of phosphatase activity in the viable hepatocyte when the combination of ligands is used. The data indicate that in the presence of caffeine and glucose together, the rate of loss of phosphorylase a is significantly increased (1.7-fold) over that in the presence of glucose alone. Phosphorylase phosphatase is activated. The sequential activation of glycogen synthase was also accelerated in the presence of both ligands. The results are consistent with an in vivo function for the nucleoside site, similar to that of glucose. A controlling role for phosphorylase in the regulation of glycogen metabolism by glucose is supported. Although the existence and nature of an intracellular effector is as yet unknown, crystallographic analyses of phosphorylase a crystals soaked in perchloric acid extracts of liver demonstrate that the negative effector site binds a natural metabolite.
Vesicle surface charge and polylysine modification of ultraviolet absorption by the olefinic bonds in charged lipid
Authors: Pawagi Asha B. , Campbell Ian M.
Published: December 2009

The results of electrical conductivity and ultraviolet absorption studies on bilayer vesicles, composed of various mixtures of saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated bovine brain phosphatidylserine (PS), in the presence and absence of polylysine indicate the following, (i) The two kinds of lipid maintain a strong transbilayer segregation over a wide range of proportions. The charged lipid (PS) preferentially locates in the inner, at PS proportions of 33% or less, and in the outer layer of the membranes when its proportion is increased to 50%. (ii) At PS proportions of 33% or less, where DPPC is the major component of the outer layer, the ability of polylysine to modify ultraviolet absorption by the olefinic bonds in the PS depends on the fluidity of the phosphatidylcholine. At higher PS proportions, where PS preferentially locates in the outer layer, modification of the ultraviolet absorption spectrum of the lipid by polylysine occurs only when the surface charge of the vesicles is diminished by the addition of bivalent metal ions. (iii) The polylysine-induced change in the ultraviolet absorption spectrum of the olefinic bonds in the vesicles was found to be very similar to that induced by dispersing unsaturated fatty acids in water rather than dissolving them in a nonpolar solvent. This suggests that polylysine modification of the vesicle spectrum may be the result of deep hydrophobic penetration by the polypeptide causing hydration and (or) parallel alignment of the dipole moments of the absorbing chromophores.
Purification and some properties of the hemolytic toxin aerolysin
Authors: Buckley J. Thomas , Halasa L. Nicole , Lund Karen D. , MacIntyre Sheila
Published: December 2009

Aerolysin, the hemolytic toxin produced by Aeromonas hydrophila, has been purified by a combination of salt fractionation, gel filtration, and ion-exchange and hydroxyapatite chromatography. The resulting protein has a molecular weight of 51 500 and appears homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. It is free of detectable protease and phospholipase activities. The purified protein can be separated into two active components with pIs of 5.39 and 5.46 by isoelectric focusing. Both components are found in the original culture supernatant indicating that the multiplicity is not due to proteolysis during isolation. Purified aeroiysin is unstable even at 25 °C and its hemolytic action is inhibited by certain reducing agents including ferrous iron and cysteine. It appears to be the only toxin hemolytic to human cells that is produced by A. hydrophila under the conditions described.
Uptake of cholesterol from the very low density lipoprotein or its remnants by the perfused rat liver
Authors: Noël Simon-Pierre , Rubinstein David
Published: December 2009

[3H]Cholesterol labelled very low density lipoproteins ([3H]chol-VLDL) were prepared to study the hepatic uptake of cholesterol associated with VLDL and its remnants in the perfused liver system. [3H]Chol-VLDL was incubated with rat postheparin plasma to produce labelled remnants in vitro. The degree of lipolysis of [3H]chol-VLDL depended on the ratio of triacylglycerols to lipase in the incubation medium. Therefore, the produced remnant of d < 1.019 g∙mL−1 had a variable lipid composition depending on the degree of lipolysis. [3H]Chol-VLDL or its remnants were added to liver perfusate and the radioactivity remaining in the perfusate was measured. The kinetic disappearance of [3H]chol-VLDL and its remnants in the perfused liver system indicated that remnant of d < 1.019 g∙mL−1 was taken up by the liver faster than the original VLDL preparation (t1/2 of 8 min vs. 51 min). Appearance of the label in bile during the perfusion was significantly faster when livers were perfused with [3H]chol-VLDL remnants as opposed to uncatabolized [3H]chol-VLDL.The results indicate that first of all, VLDL remnants produced in vitro and reisolated at density less than 1.019 g∙mL−1 do not have a fixed lipid composition but a rather variable one depending on the degree of lipolysis. Secondly, the rat liver may preferentially recognize this VLDL remnant of d < 1.019 g∙mL−1 and take it up more readily than uncatabolized VLDL. Finally when equimolar amount of cholesterol from VLDL or VLDL remnants are circulated in the liver perfusion, the VLDL remnants convey a significantly greater mass of cholesterol to the bile.
Effect of Δ9 tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena
Authors: Zimmerman Selma , Zimmerman Arthur M. , Laurence Helen
Published: December 2009

Cyclic nucleotide levels were determined in division-synchronized Tetrahymena and the effect of Δ9-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non-drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of cAMP was statistically significant; however the variation in the level of cGMP was not statistically significant.THC caused a lowering of cAMP and cGMP levels throughout the 4-h experimental treatment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8–15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (110–120 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is discussed in relation to delays in the division schedule.Nous avons déterminé le taux des nucléotides cycliques chez Tetrahymena se divisant de façon synchrone et nous avons étudié l'effet du Δ9-tétrahydrocannabinol (THC) sur le taux de ces nucléotides cycliques. Dans les cellules non traitées, se divisant de façon synchrone, il n'existe aucune variation statistiquement significative dans les teneurs du cAMP et du cGMP durant la période G2 précédant la première division. Durant le cycle cellulaire sans division (l'intervalle de temps entre la première et la seconde division synchrone), la teneur du cAMP augmente de deux fois, une augmentation statistiquement significative; cependant, la variation du taux du cGMP n'est pas statistiquement significative.
Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli
Authors: Helfman William B. , Hendler Sheldon S. , Smith Douglas W.
Published: December 2009

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or Co2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5′-triphosphates or deoxyribonucleoside 5′-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, the dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.