bluuebunny/crossref_metadata_2025 huggingface dataset:
A new mitogenic D-galactosephilic lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins
Authors: Gilboa-Garber N. , Mizrahi L.
Published: December 2009

The lectin of Erythrina corallodendron (Caesalpiniaceae) seeds was purified by heating, ammonium sulfate fractionation, and affinity chromatography on acid-treated Sepharose. The purified lectin is similar to the soybean lectin in being a glycoprotein of molecular weight around 110 000 – 120 000 and having D-galactosephilic activity. This lectin, like the soybean and Pseudomonas aeruginosa lectins, binds to D-galactosamine, N-acetyl-D-galactosamine, α- and β-D-galactosides as well as to D-galactose. Like these lectins it adsorbs onto either untreated or enzyme (papain or neuraminidase) treated human red blood cells, but exhibits a considerable mitogenic activity towards human lymphocytes (predominantly T cells) only after their treatment with neuraminidase. This mitogenic stimulation of lymphocytes is inhibited by D-galactose and its derivatives. Despite the great similarity between them, the E. corallodendron, soybean, and Pseudomonas lectins differ in regard to the intensity of their agglutinating activity towards erythrocytes obtained from different animals and human donors of diverse ABO blood groups. This phenomenon may be attributed to the difference in the affinities of the three lectins to the various D-galactose derivatives and to their molecular properties.
Inhibition of initiation of protein synthesis by tosyl-L-lysyl chloromethyl ketone
Authors: Mbikay Majambu , Garrick Michael D.
Published: December 2009

Tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits protein synthesis in intact cells and lysates. Its presence leads to polyribosome disaggregation. This inhibition is probably at the level of chain initiation because it does not slow poly(U)-dependent elongation of poly(F). This drug activates an inhibitor similar to the heme-controlled repressor in the postribosomal supernatant. High concentrations of ATP and GTP affect inhibitions of translation by the heme-controlled repressor and TLCK in a similar fashion. The latter also interferes with formation of the 40S initiation complex. Its action on translation can be prevented by preliminary incubation with thiols.
Interactions of CF1, the coupling factor of photophosphorylations with chloroplast lipids. Monolayers studies
Authors: Dijkmans Huguette , Monger Gaëtan , Aghion Jacques , Leblanc Roger M.
Published: December 2009

Surface pressure and surface potential of monolayers at a water–air interface show no polar interactions between the chloroplast total lipids and the polar protein CF1. The two components are perfectly miscible in mixed monolayers. It follows that CF1 interacts with the hydrophobic parts of the lipid molecules and that, in vivo, CF1 could penetrate in the hydrophobic core of the membranes.
Stabilization of microtubule protein in glycerol solutions
Authors: Keates R. A. B.
Published: December 2009

The consequences of short-term storage of microtubule protein at 0 °C have been examined and the ability of glycerol to prevent loss of activity has been evaluated. Three forms of activity monitored include colchicine-binding activity, critical concentration, and relative polymerizing activity. Colchicine-binding activity decayed continuously and glycerol increased the half-life roughly in proportion to its concentration (t1/2 = 57 days in 4 M glycerol). Critical concentration remained relatively constant for 2–3 days and then increased rapidly in the absence of glycerol. This rapid increase was delayed in glycerol concentrations above 1 M. Relative polymerizing activity was based in this work on the slope of the critical concentration plot. In most cases, loss of activity was apparent after as little as 24 h. This was the first observed alteration in polymerization properties of the microtubule protein and glycerol concentrations below 3 M had little effect in slowing the decay. Degradation of microtubule-associated proteins may account for these changes in polymerization properties that occurred more rapidly than tubulin denaturation. Evidence for degradation was demonstrated by sodium dodecyl sulfate gel electrophoresis. Higher glycerol concentrations slowed this degradation.
Inhibition of microtubule polymerization by the tubulin–colchicines complex; inhibition of spontaneous assembly
Authors: Keates R. A. B. , Mason G. B.
Published: December 2009

The inhibition of microtubule polymerization by colchicine requires the formation of tubulin–colchicine complexes, and inhibition of polymerization is proportional to the concentration of tubulin–colchicine complexes rather than to the total concentration of colchicine. Because the formation of such complexes is slow relative to polymerization, the kinetics of complex formation obscure the kinetics of inhibition of polymerization. We have taken defined quantities of preformed tubulin–colchicine complexes, relying on their slow dissociation, and added these to microtubule protein, which was allowed to polymerize by temperature shift to 37 °C. The degree of polymerization was then determined by measurement of turbidity at 400 nm. An appropriate kinetic analysis allowed us to distinguish effects of inhibitor on initiation and elongation phases of polymerization, without resorting to the use of initiation inhibitors. The results are consistent with a reversible association of tubulin–colchicine complex with microtubule ends blocking further elongation (Ki = 0.16 μM). Steady-state measurements suggest that copolymerization of tubulin–colchicine complex is a minor factor under the conditions used. By contrast, little inhibition of initiation was observed, possibly because tubulin–colchicine complex competes with the tubulin dimer, but not with the larger oligomers required for the initiation process.
Hydrolysis of triolein, cholesterol oleate, and 4-methylumbelliferyl stearate by acid and neutral ester hydrolases (lipases) from pigeon adipose tissues: effect of cAMP-dependent protein kinase
Authors: Severson David L. , Fletcher Thea , Groves Gerald , Hurley Brenda , Sloan Shellie
Published: December 2009

The properties of ester hydrolases (lipases) in a pH 5.2 precipitate fraction from pigeon adipose tissue have been determined in assays which have used a variety of different substrate preparations. Hydrolase activity measured with an ethanolic triolein substrate dispersion was characterized as having a single pH optimum of 7.5. In contrast, assays performed with a glycerol-dispersed triolein preparation resulted in a distinct shoulder of hydrolase activity at acid pH values in addition to a pH optimum of 7.5; addition of lecithin to the glycerol- dispersed triolein substrate preparation decreased hydrolase activity at neutral and alkaline pH values but allowed a distinct acid pH optimum (at pH 5) to be observed. Assays with glycerol-dispersed preparations of cholesterol oleate and the fluorogenic substrate, 4-methylumbelliferyl stearate (MU-stearate) (both containing lecithin) also demonstrated hydrolase activity with both acid (pH 4.5) and neutral (pH 7.5–8) pH optima. Preincubation of the pigeon adipose tissue pH 5.2 precipitate fraction with Mg2+, ATP, and cAMP resulted in a time-dependent increase in triglyceride (TG) hydrolase activity determined at pH 7 with an ethanolic triolein emulsion. This cAMP-dependent activation could be blocked by the addition of skeletal muscle protein kinase inhibitor; the addition of exogenous protein kinase (PrK) could reverse this inhibition. A Mg2+-dependent deactivation of PrK-activated TG hydrolase was observed; the rate of deactivation was enhanced by the addition of an exogenous phosphoprotein phosphatase. A cAMP-dependent PrK-catalyzed activation of hydrolase activity measured at pH 7 could also be determined with glycerol-dispersed substrate preparations of triolein, cholesterol oleate, and MU-stearate. Acid hydrolase activity (measured at pH 4.5–5 with glycerol-dispersed substrates) was not increased by preincubation with ATP, cAMP, and PrK. In experiments with glycerol-dispersed substrates, the kinetic mechanism associated with activation of pigeon adipose tissue hydrolase(s) was found to be due to an increase in Vmax with little or no change in substrate affinity.
The incorporation of choline into disaturated phosphatidylcholine in the microsomal, lamellar body, and surfactant fractions of hamster lung tissue
Authors: Delahunty Thomas J. , Johnston John M.
Published: December 2009

The specific activity of disaturated phosphatidylcholine in microsomes and lamellar bodies prepared from hamster lung tissue and in surfactant obtained by lung lavage was determined at various times following the intraperitoneal administration of [Me-3H]choline. The highest specific activity of disaturated phosphatidylcholine in the lung microsomes was attained 1 h after the administration of [3H]choline; thereafter, the specific activity declined. The specific activity of disaturated phosphatidylcholine in lamellar bodies increased steadily for 12 h after [3H]choline administration. The specific activity in lamellar bodies after 12 h exceeded the maximum specific activity achieved in the microsomal fraction (p < 0.005). The specific activity of the disaturated phosphatidylcholine in the alveolar lavage increased after an initial lag period of approximately 3 h, attaining the same specific activity as that of the lamellar bodies at the 12-h time point. The reported results are discussed in relation to the biosynthesis, storage, and secretion of the disaturated phosphatidylcholine associated with the lipoprotein, surfactant.
Fatty acid reductase in bioluminescent bacteria. Resolution from aldehyde reductases and characterization of the aldehyde product
Authors: Riendeau Denis , Meighen Edward
Published: December 2009

Fatty acid reductase from the bioluminescent bacterium Photobacterium phosphoreum, has been partially purified free of aldehyde reductase activity and with a low endogenous fatty acid content permitting the characterization of the aldehyde product of the reaction. Two aldehyde reductases, both dependent on NADH, were separated by anion-exchange chromatography from the fatty acid reductase activity. The partially purified fatty acid reductase catalyzed the synthesis exclusively of long chain aldehydes from fatty acids in the presence of ATP and NADPH as demonstrated by the conversion of [3H]tetradecanoic acid to [3H]aldehyde. Comparison of the amount of [3H]aldehyde produced with the bioluminescence responses of luciferase to the aldehyde product and standard aldehydes, both with respect to maximum light intensity and luminescent decay, established that tetradecanoic acid had been converted to tetradecanal, the aldehyde of the same chain length. These results are consistent with a mechanism involving activation of the fatty acid with ATP followed by reduction of a fatty acyl intermediate to the corresponding aldehyde.
Hybrid aspartate transcarbamoylase containing cross-linked subunits
Authors: Chan William W.-C. , Enns Caroline A.
Published: December 2009

The role of conformational changes and subunit interactions in the allosteric mechanism of aspartate transcarbamoylase was evaluated by studying hybrid enzyme molecules containing cross-linked subunits. Native enzyme was cross-linked with tartryl diazide in the presence and absence of substrate analogues. The two types of modified enzyme derivatives were each dissociated into catalytic (c3) and regulatory (r2) subunits. Hybrids were constructed with modified catalytic subunits and unmodified regulatory subunits or vice versa. Subunits from different derivatives also formed hybrids.All hybrids containing cross-linked catalytic subunits showed hyperbolic substrate saturation curves while cross-linking in the regulatory subunit alone did not abolish cooperativity. The type of cross-linking in the catalytic subunit had a decisive influence on the substrate affinity of the hybrid as well as its response to the allosteric effectors ATP and CTP. However many effects were also dependent on the presence of regulatory subunits. The results implicate a substantial conformational change in the catalytic subunit upon substrate binding and suggest an important role for the c–r interaction in the allosteric mechanism.
The phosphatidyl-myo-inositol-4,5-bisphosphate phosphatase from Crithidia fasciculata
Authors: Palmer F. B. St. C.
Published: December 2009

The phosphatase which specifically removes one phosphate group from phosphatidyl-myo-inositol 4,5-bisphosphate was purified up to 6000-fold from the cytosol of the protozoan Crithidia fasciculata. Lipoproteins which interfere with the purification were precipitated by reducing the pH to 4.5. The enzyme was isolated from the supernatant by ammonium sulfate fractionation, gel filtration (Sepharose CL-6B), ion–exchange chromatography (DEAE-Sepharose CL-6B), and hydrophobic chromatography on detergent-saturated phenyl-Sepha-rose CL-4B. The preparations had specific activities of 44–110 μmol∙min−1∙mg protein−1 with phosphatidyl-myo-inositol 4,5-bisphosphate, but were inactive with a variety of lipid and nonlipid phosphate esters. The enzyme was stable in the presence of salt and exhibited a relative mass of 117 000. It formed larger aggregates in the absence of salt and was dissociated into monomers of relative mass 57 000 by sodium dodecyl sulfate.Addition of Triton X-100 to the assay mixture reduced the dependence upon moderation of the charge of the substrate by cetyltrimethylammonium bromide. In the presence of both detergents the Mg2+ dependence of the enzyme was reduced (Km for Mg2+ = 40 μM) while the "apparent" Km for the substrate was unchanged at 240 μM. Substrate precipitation at higher Mg2+ concentrations was eliminated.
A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554
Authors: Suzuki Isamu , Kwok Siu-Chun
Published: December 2009

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.
Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate
Authors: Casola Paul G. , Possmayer Fred
Published: December 2009

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5–10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10–20 min), the rates were comparable.
The optical properties of α2-macroglobulin from normal and from cystic fibrosis plasma
Authors: Richman Janice B. Y. , Verpoorte Jacob A.
Published: December 2009

The α2-macroglobulin (α2-M) was purified from the plasma of normal individuals and from that of cystic fibrosis patients. The proteins exhibited identical optical properties. Both proteins have an absorbance coefficient of A = 1060 g∙cm−2 at 280 nm. The circular dichroism spectra are identical and indicate about 45% β-sheet structure and almost no α-helix. The spectra of solutions at pH 8.0 do not change when trypsin is added.The fluorescence spectra of the α2-M measured at pH 8.0 have contributions by tyrosine and tryptophan residues. The fluorescence intensities are identical and are enhanced about 30% when trypsin is added in 2:1 molar ratios.
The specific capsular polysaccharide of Streptococcus pneumoniae type 9V
Authors: Perry Malcolm B. , Daoust Virginia , Carlo Dennis J.
Published: December 2009

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9V (American type 68) is composed of D-glucuronic acid (1 part), D-galactose (1 part), 2-acetamido-2-deoxy-D-mannose (1 part), D-glucose (2 parts), and O-acetyl (1.6 parts). Methylation, periodate oxidation, optical rotation, and nuclear magnetic resonance studies, and partial hydrolysis showed that the polysaccharide is an unbranched high molecular weight linear polymer of a partially O-acetylated pentasaccharide repeating unit having the structure indicated below.[Formula: see text]
Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis
Authors: Hoffman Dennis R. , Haning Judy A. , Cornatzer W. E.
Published: December 2009

To produce a severe choline–methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.
Tissue distribution of sulfolipids in the rat. Restricted location of sulfatoxygalactosylacylalkylglyerol
Authors: Lingwood Clifford , Hay Genevieve , Schachter Harry
Published: December 2009

Eleven rat tissues (excluding brain) have been assayed for their ability to synthesize sulfatoxygalactosylacylalkylglycerol (SGG) from Na235SO4in vivo. These tissues were also assayed for the presence of SGG by an indirect immunofluorescence procedure using rabbit anti-SGG and frozen tissue sections. By both procedures SGG was found to be restricted to the testis; several novel sulfolipids were detected during this study.
Effects of Intravenous versus intragastric glucose pretreatment on triglyceride secretion by perfused livers of fed rats
Authors: Semple D. J. , Wolfe B. M.
Published: December 2009

The effects of the route of administration of 450 mg [U-14C]glucose to 250-g fed rats in vivo on the subsequent release of triglycerides from the perfused liver was studied. Livers were perfused for 180 min using a nonrecycling medium containing 10 mM glucose, 2 mM lactate, 0.2 mM pyruvate, and 100 μU/mL porcine insulin. Biopsies were obtained at the beginning and end of the perfusions. The perfusate was collected and the secreted triglycerides were analyzed.Slower absorption of the intragastric glucose load contrasted with the rapid entry of the intravenous load; however, the total liver counts were not significantly affected by the route of glucose delivery. Hepatic glycogen concentration was also not significantly different, but the percent of total liver counts which was present in glycogen was significantly higher after intravenous glucose. The majority of the radioactivity in the livers of both groups of rats was present in water-soluble metabolites, with lesser amounts in triglycerides and phospholipids. Radioactivity in hepatic triglycerides declined significantly during perfusion only in the rats which had received glucose intravenously. The mean rate of triglyceride secretion from the livers of rats receiving the glucose intravenously was significantly lower than that of the rats receiving glucose intragastrically (0.21 ± 0.05 vs. 0.97 ± 0.28 mg/g liver per 180 min, p < 0.05). The route of glucose administration affects both entry of glucose into the blood and subsequent hepatic triglyceride metabolism and secretion.
Reverse cholesterol transport from fibroblasts to high density lipoproteins: computer solutions of a kinetic model
Authors: Verdery Roy B.
Published: December 2009

A model for cholesterol balance between fibroblasts and high density lipoproteins which includes first steps in cholesterol egress is proposed. Kinetic solutions of this model, obtained by digital computer integration of associated differential equations, showed that 15-fold variations in high density lipoprotein levels might be expected to cause less than 8% change in cell cholesterol and twofold variations in lecithin:cholesterol acyltransferase activity might be expected to cause less than 10% change in cell cholesterol. These theoretical calculations are limited by deficiencies in the model. However, they can be used as adjuncts to direct experiment to evaluate the hypothesis that high density lipoproteins and lecithin:cholesterol acyltransferase are involved in cholesterol removal from peripheral cells.
The relationship between variations in levels of serotonin acetyltransferase activity and cGMP content in cultured chick pineal glands
Authors: Wainwright S. D. , Wainwright Lillian K.
Published: December 2009

Chick pineal glands cultured in continuous darkness exhibited a circadian rhythm in 3′,5′-cyclic GMP (cGMP) content. cGMP content increased earlier and fell later than in glands incubated under diurnal lighting. A "spike" of further increase in cGMP content preceded start of decline in acetyl-CoA:arylamine N-acetyltransferase (NAT) activity.Theophylline or 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (compound Ro 20.1724) alone markedly stimulated increase of NAT activity in the dark, and retarded subsequent loss of activity either in the dark or in the photoperiod of a diurnal cycle of illumination. In constant darkness a combination of the two agents elicited greater increases of NAT than either alone and suppressed subsequent loss of activity for several hours. Under diurnal lighting conditions the combination was only slightly more effective than either alone. Compound Ro 20.1724 suppressed the precipitous loss of NAT activity seen when glands are transferred prematurely to the light. However, neither the rate or extent of loss of activity was greatly affected by theophylline.Effects of theophylline and compound Ro 20.1724 upon levels of NAT activity appeared to be due primarily to effects upon the process(es) by which enzyme activity is lost. They could not be attributed solely to effects upon pineal contents of cGMP and (or) 3',5'-cyclic AMP (cAMP). Theophylline alone was as effective as the drug combination in eliciting and maintaining elevated pineal content of cGMP, but had little effect upon cAMP content. Compound Ro 20.1724 alone elicited large increases in pineal contents of both nucleotides but did not sustain them.
Cholesteryl sulfate; measurement with β-sitosteryl sulfate as an internal standard
Authors: Huang Y. S. , Eid K. , Davignon J.
Published: December 2009

A new method is described for the measurement of cholesteryl sulfate (CS) in plasma. Taking advantage of the use of β-sitosteryl sulfate (SS) as an internal standard it is simpler and more rapid than the methods currently in use. It does not require the use of radioactive isotopes. A potential contamination of the CS band by free cholesterol is excluded by developing the thin-layer chromatography plate on which the sterols are first separated with a second solvent system which pushes free cholesterol to the solvent front without appreciably moving the CS band. The method is sensitive and reproducible and may be advantageous for the screening of large populations. The values obtained for plasma CS concentrations in normal subjects compare well with those measured by more elaborate techniques.
Differential ontogeny of estrogen responsiveness in the chick embryo liver
Authors: Elbrecht A. , Williams D. L. , Blue M.-L. , Lazier C. B.
Published: December 2009

The development of estrogen responsiveness in embryonic chicken liver has been studied in terms of specific genomic responses to the hormone. Embryonated eggs were injected with estradiol at various stages of development, and after 48 h the hepatic synthesis of the apoproteins II and B of very low density lipoprotein (VLDL) was determined by incubation of liver slices with [3H]leucine and analysis of the tissue supernatant by specific immunoprecipitation. Significant estrogen induction of synthesis of apoprotein II of VLDL could be seen by the 11th day of embryonic development. The induction of apoprotein B, the other major apolipoprotein of VLDL, paralleled that of apoprotein II. It was reported earlier that the synthesis of vitellogenin did not become estrogen inducible until about the 15th day of embryonic development. The dissociation of the vitellogenin and apoprotein B responses can be confirmed by direct electrophoretic analysis of [3H]serine-labelled liver homogenates. The results are discussed in relation to the ontogeny of the estrogen receptor and the possible involvement of other controlling factors.
Effects of dietary protein on composition and turnover of apoproteins in plasma lipoproteins of rabbits
Authors: Roberts D. C. K. , Stalmach M. E. , Khalil M. W. , Hutchinson J. C. , Carroll K. K.
Published: December 2009

The hypercholesterolemia and atherosclerosis produced in rabbits by feeding cholesterol-free, semipurified diets are due to the use of casein as the protein component of such diets and can be prevented by replacing the casein with isolated soy protein. To investigate the reasons for the differing effects of these dietary proteins on plasma cholesterol levels, plasma lipoproteins were isolated from rabbits fed semipurified diets containing either casein or isolated soy protein, labeled with 125I, and reinjected into rabbits fed one or other of these two diets. 125I-labeled apoproteins of intermediate density lipoprotein, isolated from rabbits on either diet, turned over more rapidly in rabbits fed soy protein compared with those fed casein. 125I-labeled apoproteins of very low density lipoprotein from rabbits fed soy protein were transferred to high density lipoprotein more rapidly than those from rabbits fed casein. In this case, the results were determined primarily by the diet fed to the donor rabbits, but the diet fed to the recipients also appeared to have some influence. The apoproteins of plasma lipoproteins from rabbits fed casein or soy protein were separated by isoelectric focussing and tentatively identified by comparison of their isoelectric points with those of apoproteins from human plasma lipoproteins. The concentration of apoprotein E was markedly increased in the very low density and intermediate density lipoproteins of casein-fed rabbits, and apoprotein C was also increased in the very low density lipoprotein of rabbits fed casein, compared with those fed soy protein. Effects of dietary proteins on plasma cholesterol may be secondary to their effects on the composition and metabolism of the protein components of plasma lipoproteins.
Exchange of free cholesterol between low density lipoproteins and human adipocytes
Authors: Angel A. , Yuen R. , Nettleton J. A.
Published: December 2009

Free cholesterol uptake from low density lipoproteins (LDL) by fat cells was investigated using exchange-labelled [1,2-3H]cholesterol–LDL and isolated human adipocytes, prepared by collagenase digestion of subcutaneous fat tissue biopsies. Radioactivity appeared in the cellular lipids within 2 min and accumulated at a slower rate approaching linearity for 3 h. At 5 and 120 min, 90% of the recovered cellular radioactivity was found in the bulk lipid fraction and the remainder was distributed among organelle fractions. Uptake of [3H]cholesterol was linearly related to substrate concentrations up to 5 μg unesterified cholesterol – LDL∙mL−1. At higher concentrations up to 22 μg unesterified cholesterol – LDL∙mL−1, [1,2-3H]cholesterol accumulated more gradually but did not reach a plateau. Metabolic inhibitors, sodium azide plus sodium fluoride, had no effect on radiocholesterol uptake. Native LDL and high density lipoprotein at equivalent unesterified cholesterol concentrations decreased [1,2-3H]cholesterol uptake, to a similar extent suggesting isotope dilution. The results demonstrate a rapid, receptor-independent uptake of cholesterol from LDL by adipocytes, consistent with an exchange mechanism. This process could explain the rapid labelling of adipose tissue cholesterol in vivo following intravenous administration of radiocholesterol and efflux of free cholesterol from adipose tissue during starvation.
Secretion of nascent lipoproteins by isolated rat hepatocytes
Authors: Krul Elaine S. , Dolphin Peter J. , Rubinstein David
Published: December 2009

The nature of the nascent lipoproteins secreted by suspensions of isolated rat hepatocytes incubated in a lipid-deficient medium was investigated. Samples of the concentrated medium after 12 and 24 h of incubation were resolved by gel filtration and demonstrated that lipoproteins were secreted with a wide spectrum of particle sizes. Particles corresponding to sizes of serum very low density lipoproteins (VLDL) and low density lipoproteins (LDL) had similar levels of apolipoproteins (apo) B and E as serum VLDL when determined by electroimmunoassay, suggesting that the liver cell secretes a "small" VLDL under these conditions and not an LDL particle as present in the serum. Lipid analyses of the secreted triglyceride-rich particles show them to be similar in composition to serum VLDL with the exception of their cholesterol ester content, which was much lower in the hepatocyte-secreted VLDL. Incorporation of 3H-labelled amino acids into the VLDL apoproteins from the incubation medium after 24 h was determined after ultracentrifugal isolation (d < 1.063 g∙mL−1) and urea–gel electrophoresis, and found to be 70% and 22% of the total applied radioactivity for apo B and apo E, respectively. The lack of immunochemicaily detectable apo C-II and C-III in the isolated nascent VLDL and the lack of significant radioactive incorporation confirmed their visual absence from the gels. Further purification of the VLDL apo E by immunoaffinity chromatography showed it to consist of two narrowly separated bands on 7 M urea – polyacrylamide gels. Apo B was secreted only with particles having mean diameters of greater than 194 Å. In contrast, 75% of the total secreted apo E was associated with fractions of smaller particle diameters. This apo E (LpE) was almost equally distributed in two peaks corresponding to a particle size of approximately 100 Å and a molecular weight of < 60 000, respectively. Only 35% of the total apo E was found in the comparable fractions when hepatocytes from hypercholesterolemic rats were used. Thus, normal hepatocytes secrete a significant proportion of apo E, as a low molecular weight, essentially lipid-free form. The apparent secretion rates, for the apoproteins (mean ± SEM) for hepatocytes from normal rats, were 77.0 + 10.6 μg∙h−1∙g cell protein−1 (apo B) and 71.7 ± 8.6 μg∙h−1∙g cell protein−1 (apo E) after 24 h.
Composition and metabolism of very low density lipoproteins in dog cardiaclymph
Authors: Julien P. , Angel A.
Published: December 2009

In the present study, very low density lipoprotein (VLDL, d < 1.006) in cardiac lymph was characterized to determine its role as a metabolic substrate in the interstitial compartment. A major efferent cardiac lymph trunk was cannulated in fasting (18 h) dogs (20–27 kg). Three to five millilitres of lymph were collected over 3–4 h at 4 °C. Cardiac lymph VLDL concentration was 1.7 ± 0.7 mg protein∙100 mL−1 compared with 1.8 ± 0.8 mg protein∙100 mL−1 in plasma. The VLDL triglyceride concentration in lymph was 1.0 ± 0.3 mg triglyceride∙100 mL−1 with triglyceride/protein ratio of 0.9 compared with plasma VLDL triglyceride of 5.0 ± 1.6 mg∙100 mL−1 with a triglyceride/protein ratio of 5.5. Electron microscopy of VLDL revealed globular particles with a mean diameter of 388 Å in lymph and 661 Å in plasma. Thus, cardiac lymph VLDL are smaller and contain less triglyceride per particle than plasma VLDL. Following i.v. administration of human 125I-labelled low density lipoprotein ([125I]LDL, d 1.025–1.045), cardiac lymph/plasma LDL specific activity ratio was 0.52 ± 0.15 (n = 3) and 0.55 ± 0.15 (n = 4) at 3 and 27 h, respectively. The fact that the specific activity ratio did not reach 1 at plateau suggests continuous addition of unlabelled LDL in the cardiac interstitium, presumably from VLDL precursors. These findings demonstrate that on a protein basis the concentration of VLDL in cardiac lymph equals that of plasma, and also suggests that VLDL degradation and LDL production occur in the cardiac interstitial space.
Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation
Authors: Hunting Darel , Henderson J. Frank
Published: December 2009

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2′-dAMP to inhibit the 3′ → 5′ exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.
Regulation of the activity of a calcium-activated neutral protease during differentiation of skeletal myoblasts
Authors: Kaur Harjeet , Sanwal Bishnu D.
Published: December 2009

A calcium-activated neutral protease activity appears concomitantly with myotube formation during the differentiation of a cell line of rat skeletal myoblasts. Other proteases such as cathepsin D and plasminogen activator, however, do not show any changes in their activities. The appearance of the protease is not fusion dependent, as judged by assays of fusion defective myoblast mutants. The formation of the protease is suppressed along with differentiation in the presence of 5-bromodeoxyuridine. Undifferentiated myoblasts contain a potent inhibitor of the protease. The inhibitor, which is probably proteinaceous in nature, is lost during the differentiation of the cells into myotubes. This mode of regulation of an enzyme during differentiation seems so far to be an unique example of its kind.
Mg2+ and Mn2+ effects on membrane-bound and detergent-solubilized adenylate cyclase
Authors: Drummond George I.
Published: December 2009

Mg2+ and Mn2+ stimulation of basal, F−, and guanyl-5′-yl imidodiphosphate (GPP(NH)P) stimulated adenylate cyclase in particulate and detergent-solubilized preparations of skeletal muscle, cardiac muscle, and erythrocytes was examined. Solubilization decreased the concentrations of Mg2+ required for half-maximal velocity and for saturation. Concentrations of Mn2+ required for saturation and for half-maximal velocity of particulate preparations was much lower than for Mg2+ and these values were not markedly reduced by solubilization. Particulate and soluble preparations were similarly stimulated by NaF and GPP(NH)P. Activation of the heart and skeletal muscle enzyme by NaF and GPP(NH)P greatly reduced the Mg2+ requirement; this was seen with both particulate and solubilized preparations. It is suggested that solubilization removes Mg2+ action at a regulatory site; MgATP and MnATP are both effective at the catalytic site, the latter producing higher Vmax.
Regulation of enzyme activity in the hibernator. A kinetic and spectroscopic study of muscle pyruvate kinase from the Arctic ground squirrel
Authors: Morse George A. , Behrisch Hans W.
Published: December 2009

Pyruvate kinase skeletal muscle of the Arctic ground squirrel was purified to homogeneity. The purified enzyme variants from the summer-active and winter hibernating squirrel appear to be identical with a near-neutral pI of 6.9 and a molecular weight of 234 000 as determined by gel filtration chromatography on Bio-Gel A-0.5m. Evidence for subunit interaction during inhibition by L-phenylalanine is demonstrated with ultraviolet derivative spectroscopy. A model for this interaction and its importance for a regulatory role are discussed. The absence of a temperature break in the Arrhenius plot for the pyruvate kinase reaction, the kinetic and physical data, and the near-neutral pI, suggest an amino acid composition that conserves the overall geometry and resultant kinetic behavior which render regulation of the enzyme insensitive to temperature.
Enzyme induction and hepatic glycerolipid synthesis in rats treated with 3-methlcholanthrene
Authors: Goldberg D. M. , Roomi M. W. , Yu A. , Roncari D. A. K.
Published: December 2009

Fasting male rats fed 3-methylcholanthrene in a daily dose of 40 mg∙kg body weight−1 gave evidence of hepatic microsomal enzyme induction after 3 days through significantly increased hepatic aryl hydrocarbon hydroxylase activity, cytochrome P448 content, and characteristic changes in microsomal proteins analysed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Concomitantly, activities of aminopyrine N-demethylase and microsomal γ-glutamyltransferase, which are increased in phenobarbital-treated rats, significantly declined. In contrast to phenobarbital, which has been previously shown to increase hepatic triacylglycerol content and in vitro glycerolipid synthesis, 3-methylcholanthrene did not affect hepatic triacylglycerol content, but did inhibit glycerolipid synthesis by cell-free preparations of rat liver and significantly reduced serum triacylgiycerol concentration. Thus, the two prototypical drugs inducing characteristically different changes in microsomal enzyme and hemoprotein response also seem to differ in their effect upon another important microsomal function, hepatic glycerolipid synthesis.
Differential effects of ethanol and the rpsL1 (strA1) ribosomal mutation on the synthesis of an unusual protein coded by bacteriophages R17 and MS2 RNA
Authors: Laughrea Michael
Published: December 2009

During translation of R17 and MS2 RNA, ethanol stimulates the synthesis of a coat-related protein which has identical electrophotetic mobility to that of polypeptide 7 studied by Atkins and his colleagues. Streptomycin stimulates the synthesis of this polypeptide and broadens the protein band. In contrast the ribosomal protein S12 mutation rpsL1 (strA1) has no detectable effect on its synthesis.
Temperature-induced changes in nuclear pore complex frequencies nuclear envelope surface areas and nuclear volumes in light-synchronized Euglena
Authors: Greenwood John S. , Lott John N. A.
Published: December 2009

An autotrophic culture of Euglena, synchronized using a day: night (D:N), 14:10-h cycle, was subjected to a 21.5 → 31.5 °C temperature shift and then to a reversed shift in temperature after three D:N cycles at 31.5 °C. Nuclear pore complex (NPC) number per square micrometre and nuclear surface area and volume determinations were made on G1 cells at various intervals. Cells sampled immediately prior to the 21.5 → 31.5 °C shift had a mean value of 37.68 NPC∙μm−2 nuclear envelope surface area, 30.40 NPCs/μm2 after three D:N cycles at.31.5 °C and 39.98 NPCs/μm2 after three D:N cycles at the resumed culture temperature of 21.5 °C. Thus temperature changes affect NPC numbers per square micrometre and these changes are reversible.Mean nuclear surface area was 125.76 μm2 immediately prior to the 21.5 → 31.5 °C shift, and decreased over two D:N cycles at 31.5 °C to 101.30 μm2 by the end of the third D:N cycle. Nuclear envelope surface area, one and two D:N cycles after the 31.5 → 21.5 °C shift, was approximately equal that prior to the 21.5 → 31.5 °C shift. After the third D:N cycle, however, nuclear surface area had increased to 173.05 μm2. The changes in nuclear surface area resulted in large differences in the estimates of the total number of NPCs per nucleus. Euglena immediately prior to the 21.5 → 31.5 °C temperature shift had 4739 NPCs/nucleus; immediately prior to the 31.5 → 21.5 °C shift had 3079 NPCs/nucleus; and had 6919 NPCs/nucleus at 21.5 °C and three D:N cycles after the 31.5 → 21.5 °C shift. Estimates of the number of NPCs per cubic micrometre of nuclear volume were almost identical between these samples.
Inhibition of bacterial D-3-hydroxybutyrate dehydrogenase by substrates and substrate analogues
Authors: Kluger Ronald , Tsui Wing-Cheong
Published: December 2009

D-3-Hydroxybutyrate dehydrogenase (Pseudomonas lemoignei, EC 1.1.1.30) is subject to substrate inhibition by acetoacetate at concentrations above 5 mM but not by D-3-hydroxybutyrate at concentrations up to 50 mM. NADH causes substrate inhibition at concentrations over 0.1 mM as does NAD. Kinetic analysis suggests that substrate inhibition by acetoacetate is due to its binding to enzyme lacking NADH, a consequence of the ordered bibi mechanism. Substrate inhibition by NADH and NAD arises from binding of these species to a secondary site. This is confirmed by kinetics which indicate that ADP and ATP compete with NAD and NADH at both sites.New analogues of acetoacetate were synthesized to test the specificity requirements of the acetoacetate binding site which has been proposed to contain a hydrogen bond donor and a cation spaced to receive acetoacetate. Both dimethoxyphosphinylacetate and methyl 2-methoxyphosphinylacetate fulfill the structural requirements and are effective. They thus join methyl acetonylphosphonate as the only known competitive inhibitors for the acetoacetate site, confirming the proposed structure.
Effects of altered ribonucleotide concentrations on ribonucleotide reduction in intact Chinese hamster ovary cells
Authors: Hunting Darel , Hordern Joyce , Henderson J. Frank
Published: December 2009

The effect of changes in ribonucleoside triphosphate concentrations on those of deoxyribonucleotides has been determined in Chinese hamster ovary K-1 cells. Ribonucleoside triphosphate concentrations were manipulated by the use of drugs or naturally occurring purines or pyrimidines. Changes in the concentration of each ribonucleoside triphosphate led to changes in the concentration of each corresponding deoxyribonucleoside triphosphate. The relationships between the concentrations of CTP and dCTP and between those of GTP and dGTP were linear over a wide range. The relationship between the concentrations of ATP and dATP was linear over the narrow range studied, while the relationship between UTP and dTTP concentrations was curvilinear. Overall, these results demonstrate that ribonucleotides play an important role in controlling deoxyribonucleotide concentrations but the exact mechanism of this control remains to be determined.
The acylation of 1-acyl sn-glycero-3-phosphorylcholine by glial and neuronal nuclei and derived neuronal unclear envelopes: a comparison of unclear and microsomal membranes
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

A neuronal nuclear fraction (N1), a glial nuclear fraction (N2) and a fraction containing microsomal membranes (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from fraction N1. In comparison with the parent fraction N1, fraction E had a much lower yield of protein (0.077 mg/g cerebral cortex), a low specific DNA content, an eightfold higher specific phospholipid content (0.85 μmol phospholipid phosphate/mg protein) and a very similar phospholipid distribution profile. Using 100, 50, and 25 μM 1-acyl-sn-glycero-3-phosphorylcholine (labelled with [3H]palmitate) and 100 μM oleoyl CoA, the activity of acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase was studied in vitro. Fractions N1 and N2 had specific activities which were two to three times the specific activities shown for fraction P3. Fraction E was particularly enriched in this acylation activity and had specific activities which were 6 times those of fraction N1 and 11–19 times those of fraction P3. The existence of nuclear acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase activity was indicated as was a particularly high concentration of this enzyme within the nuclear envelope. In assays of lysolecithin–lysolecithin transacylase activities, fraction N2 (glial nuclei) showed the highest specific activities, being three to four times those of fractions N1 or P3. This transacylase activity (N2) was as high as 40% of the corresponding acyltransferase activity measured in this fraction using oleoyl CoA as acyl donor.
A sensitive and convenient assay procedure for transferrin and its application to the purification of mouse transferrin
Authors: Letendre Elaine D. , Holbein Bruce E.
Published: December 2009

A sensitive procedure employing 55Fe binding was developed for the assay of transferrin. This procedure took advantage of the pH dependence and reversibility of iron binding by transferrin and was applicable to both human and mouse transferrins. Excess iron, after saturation of the transferrin iron-binding sites with 55Fe, supplied as Fe–citrate, was efficiently removed by its binding to Amberlite CG-400 anion-exchange resin.Mouse transferrin was purified from plasma using a combination of ammonium sulphate fractionation and ion-exchange chromatography. Two peaks of pure transferrin were obtained by DEAE-Sepharose chromatography. Mouse transferrin was found to have a molecular weight of 80 000.
Chloroplast deoxyribonucleic acid Isolation: purity achieved without nuclease digestion
Authors: Rhodes Patsy R. , Kung S. D.
Published: December 2009

Chloroplasts, obtained from plant tissues homogenized in liquid nitrogen, were freed of nuclei on silica sol gradients. Buoyant density analysis of denatured–renatured DNA and the clarity of restriction fragment patterns demonstrate the purity of these preparations. In this manner, chloroplast DNA free of substantial nuclear DNA contamination was obtained from several plant species without the use of a deoxyribonuclease digestion step.