bluuebunny/crossref_metadata_2025 huggingface dataset:
Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase
Authors: Riordan John R.
Published: December 2009

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 °C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and β-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPasa activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.
Subcellular distribution and partial characterization of the three major classes of concanavalin A receptors associated with rat brain synaptic junctions
Authors: Gurd James W.
Published: December 2009

Synaptic junctional complexes from rat brain contain three major classes of glycoproteins which react with concanavalin A. They have apparent molecular weights of 110 000 (GP 110) 130 000 (GP 130), and 180 000 (GP 180). They are present in postsynaptic densities but are not found in microsomes, axolemma, synaptic vesicles, or myelin and are present in low concentrations in the Triton X-100 extract obtained during the preparation of synaptic junctions suggesting that they are uniquely localized to the postsynaptic apparatus. Reaction of the individual glycoproteins, partially purified by affinity chromatography on concanavalin A – agarose followed by polyacrylamide gel electrophoresis, showed that GP 130 contained the most receptor sites for concanavalin A per unit of protein followed by GP 180 and GP 110. Of the receptor sites for concanavalin A, 60–70% were subject to hydrolysis by endoglycosidase H indicating that the lectin reacts primarily with polymannose asparagine linked oligosaccharides. Each of the glycoproteins also reacted to varying degrees with the lectins from Lotus tetragonolobus (specific for α-L-fucose), wheat germ (N′-acetyl-D-glucosamine and (or) sialic acid), and lentils (mannose, N′-acetyl-D-glucosamine). Chromatography of 125I-labelled concanavalin A positive glycoproteins on wheat germ Sepharose resolved GP 110 and GP 180 into wheat germ positive and negative components indicating the presence of some structural heterogeneity within these molecular weight classes.
Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine
Authors: Boulanger Yvan , Schreier Shirley , Leitch Leonard C. , Smith Ian C. P.
Published: December 2009

Anesthetics bound to model membranes were observed directly by means of deuterium nuclear magnetic resonance (NMR). The specifically deuterated local anesthetics procaine and tetracaine were synthesized, and their partition coefficients (water:phosphatidylcholine) and pKa values determined. The interaction of these anesthetics with lamellar dispersions of egg phosphatidylcholine was studied by 2H nuclear magnetic resonance and by electron spin resonance (ESR) of a spin-labelled phospholipid at low (5.5) and high (9.5) pH. The ESR experiments suggest that tetracaine intercalates in the membrane and that it equilibrates between water and the phospholipid bilayers of the multilamellar system. The NMR results are consistent with a model where the anesthetic is (I) free in water, (2) weakly bound, and (3) strongly bound to the membrane. A fast exchange exists between the two first sites, but exchange is slow with the third site. Binding of type 3 is observed only at high pH for procaine, whereas it is found both at low and high pH for tetracaine. Calculations of the partition coefficients for the charged and uncharged forms of tetracaine indicate that both sites, 2 and 3, are occupied by the charged form at low pH and by the uncharged form at high pH. The partition coefficient for the weakly bound species was estimated from an analysis of the dependence of line width on the lipid to water ratio. The NMR data suggest that the binding sites for the strongly bound charged and uncharged species are different, the former probably being closer to the membrane–water interface. Estimates of molecular order parameters for the strongly bound species indicate that it is located with its long molecular axis approximately parallel to the director for ordering of the fatty acyl chains. A small increase in lipid ordering by tetracaine is observed at low pH, as evidenced by 2H NMR of the deuterated N-methyl groups of phosphatidylcholine; the reverse occurs at high pH.
Identification and characterization of outer membrane fragments released by Aeromonas sp.
Authors: MacIntyre S. , Trust T. J. , Buckley J. T.
Published: December 2009

Aeromonas hydrophila and Aeromonas salmonicida were shown to release "blebs" or fragments averaging 20–30 nm in diameter. The protein composition of the fragments was very similar to that of the corresponding outer membrane by sodium dodecyl sulphate – polyacrylamide gel electrophoresis and the fragments were shown to contain phospholipid and lipopolysaccharide. The results therefore indicate that the blebs are derived from the outer membrane of these organisms. Fragments isolated directly by differential ultracentrifugation were indistinguishable from fragments isolated by salt fractionation and gel filtration in chemical composition, protein composition, and density. However fragments isolated directly contained much less glycerophospholipid–cholesterol acyltransferase than those isolated by salt fractionation.The potential usefulness of membrane fragments to the bacteria and as a tool in the study of outer membrane structure and function is discussed.
Parallel regression of cold-induced changes in ultrastructure, composition, and properties of brown adipose tissue mitochondria during recovery of rats from acclimation to cold
Authors: Desautels Michel , Himms-Hagen Jean
Published: December 2009

When cold-acclimated rats are returned to a thermoneutral environment (28 °C) after 5 weeks at 4 °C, the size of the enlarged interscapular brown adipose tissue decreases during the 1st week. The altered composition of the mitochondria, however, reverts to normal during deacclimation more rapidly than the size of the tissue: a marked decrease in binding of purine nucleotides occurs by 3 days and a decrease in the proportion of polypeptides of molecular weight 25 000–35 000 occurs by 1 day. The very rapid decrease in the polypeptides, compared with the slower decrease in binding of purine nucleotides, known to be to a 32 000 polypeptide, suggests an initial conversion of masked binding sites to another form. Mitochondria isolated from cold-acclimated rats are larger than those of warm-acclimated rats, a difference which appears slowly during acclimation to cold, requiring 7–14 days for full development; during deacclimation the increase in size disappears by 7 days. The ultrastructure of mitochondria isolated from cold-acclimated rats shows inmerices arranged in numerous, narrow, interconnecting parallel sheets in contrast to the sparser broad tubes and bands in mitochondria isolated from warm-acclimated rats. This difference in ultrastructure develops in a biphasic manner during acclimation to cold: an initial appearance (1–12 h) of the parallel arrangement, associated with a transient increase in size, is no longer apparent at 1–3 days and reappears only after 7–14 days. During deacclimation the reversal to the tubular form requires approximately 7 days. During deacclimation the changes in brown adipose tissue mitochondrial composition, structure, size, and properties and in tissue size appear to occur independently.
The effect of amphipaths on the flavin-linked aerobic glycerol-3-pliosphate dehydrogenase from Escherichia coli
Authors: Robinson John J. , Weiner Joel H.
Published: December 2009

When assayed by the phenazine methosulphate coupled 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, the flavin-linked aerobic glycerol-3-phosphate dehydrogenase from Escherichia coli strain 7 showed a marked loss of activity if depleted of detergent, and this activity could be reconstituted with amphipaths. However, enzyme activity, as measured by the ferricyanide reduction assay, showed no loss of activity upon detergent depletion. Cross-linking studies indicated that amphipath was not inducing any significant quaternary structural change in the enzyme. Flavin fluorescence titration experiments indicated a single type of binding site for glycerol 3-phosphate whose affinity was unaffected by the presence of amphipath; apparent Kd values of 19.5 and 17.1 mM were measured in the presence and absence of amphipath, respectively. The Michaelis–Menten constants for DL-glycerol 3-phosphate, as measured by the phenazine methosulphate coupled MTT and ferricyanide reduction assays in the presence of amphipath, were found to be 1.9 and 10.2 mM, respectively. Cupric ions were found to specifically inhibit the phenazine methosulphate coupled MTT reducing activity while zinc ions inhibited the ferricyanide reducing activity. Circular dichroic studies provided initial evidence for a conformational change in the presence of the nonionic detergent Brij 58 and this was substantiated by quenching of tryptophan fluorescence at concentrations of Brij 58 equivalent to those needed to stimulate activity. We conclude from these studies that amphipaths may specifically induce a phenazine methosulphate binding site thus permitting reduction of this electron acceptor.
Stereochemical substrate requirements of lecithin:cholesterol acyltransferase and its inhibition by enantiomeric lysolecithins
Authors: Smith Norman B. , Kuksis Arnis
Published: December 2009

The suitability of 1-sn-phosphorylcholine- and 2-sn-phosphorylcholine-diacylglycerols as substrates for lecithin:cholesterol acyltransferase (LCAT) was assayed under standardized conditions using sonicated liposomes of radioactive phospholipids. The results were compared with those obtained for the corresponding molecular species of the natural 3-sn-phosphoryl-cholinediacylglycerol. It was found that with the 2-sn-phosphorylcholinediacylglycerol, LCAT had only 16% of the activity that it showed for 3-sn-phosphorylcholinediacylglycerol, while its activity with 1-sn-phosphorylcholinediacylglycerol as the substrate was not significant. It is concluded that LCAT possesses highly specific steric and positional requirements for its phosphatidylcholine substrates and that, like phospholipase A2, it has a significant activity with the 2-sn-phosphorylcholinediacylglycerol. It was also shown that the activity of LCAT is inhibited by both enantiomers of lysophosphatidylcholine as well as Triton X-100. In all instances the inhibition was reversed by the addition of lipid-free albumin. It is concluded, therefore, that the inhibition of LCAT by lysophosphatidylcholine is a detergent rather than an end-product inhibition phenomenon.
Two large glycoprotein fragments related to the NH2-terminal part of the adrenocorticotropin–β-lipotropin precursor are the end products of the maturation process in the rat pars intermedia
Authors: Crine P. , Seidah N. G. , Jeannotte L. , Chrétien M.
Published: December 2009

Pulse and pulse–chase studies of the peptide hormones synthesized in the rat pars intermedia showed that during the maturation of pro-opiomelanocortin (the adrenocorticotropin-β-lipotropin (ACTH–β-LPH) precursor) the N-terminal portion of the precursor was released as a glycopeptide of relative mass of 17 000–19 000 and did not undergo further proteolytic cleavage. This peptide is therefore proposed to be the biologically active species related to the amino terminal fragment of pro-opiomelanocortin in the rat pars intermedia.
D-Glucose and D-gluconate transport in vesicles from Pseudomonas putida
Authors: Al-Jobore Ameera , Moses G. , Taylor N. F.
Published: December 2009

Vesicles prepared from glucose-grown cells of Pseudomonas putida (ATCC, 12633) retain glucose oxidase (GOX) and gluconate dehydrogenase (GADH) activity and actively transport D-glucose, 2-deoxy-D-glucose (2DOG), 3-deoxy-3-fiuoro-D-glucose (3FG), and D-gluconate by saturable processes. The transport of these substrates is stimulated by the addition of L-malate or reduced phenazine methosulphate (PMS). Vesicles prepared from succinate-grown cells of P. putida lose their capacity to transport D-glucose, 2DOG, and 3FG by a saturable process. The transport and accumulation of D-gluconate, however, is retained with a Kx value of 65 μM and a Vmax of 1.0 nmol∙mg protein−1∙min−1. The rate of D-gluconate transport is stimulated by the addition of reduced PMS or L-malate with a reduction in the Kx value to 42.0 μM. Respirometric studies with these vesicles indicate the presence of an active GOX and L-malate dehydrogenase but a defective GADH. Thus a reductase activity is detected in the presence of D-gluconate and either 2,6-dichloroindophenol (DCIP) or ferricyanide, as measured by a decrease in absorbance at 600 and 420 nm, respectively. Measurements on these vesicles with the oxygen electrode, however, indicate that no electron transfer from GADH to oxygen occurs. This is in contrast to the results with glucose-grown vesicles or with L-malate or D-glucose as substrates in the succinate-grown vesicles. A comparison between glucose and gluconate oxidase activity in native and detergent-treated vesicles is made. The significance of these results in relation to the presence of a glucose carrier in P. putida and other pseudomonads is presented.
Control of fat cell phosphatidate phosphohydrolase by lipolytic agents
Authors: Moller F. , Wong K. H. , Green P.
Published: December 2009

The Mg2+-dependent phosphatidate phosphohydrolase activity increased in the microsomal and decreased in the soluble fraction of isolated rat fat cells incubated for short periods with the lipolytic hormones or agents, epinephrine, cyclic AMP, theophylline, and dibutyryl cyclic AMP. Adrenocorticotropin, on the other hand, increased not only the microsomal but also the soluble activity. The increases in microsomal activity ranged from 30 to 134% with epinephrine to almost 200% with dibutyryl cyclic AMP. The decreases in soluble activity were more modest. The effect of epinephrine was inhibited by the β-adrenergic antagonist propranolol while the α-antagonist phentolamine enhanced it. These results strongly suggest that the fat cell phosphatidate phosphohydrolase is controlled through the β-adrenergic receptor and the activity of adenylate cyclase. Lipolysis, as measured by fatty acid release, was stimulated in a similar pattern as the microsomal activity suggesting parallel activation of the hormone sensitive lipase and phosphatidate phosphohydrolase. It is speculated that the activation of this lipogenic enzyme by lipolytic stimuli may represent a mechanism whereby fatty acid release from adipose tissue may be modulated and intracellular fatty acid accumulation maybe counteracted during accelerated lipolysis in adipose tissue.
Changes in liver cell plasma membrane polypeptides of phalloidin-treated rats with special reference to the actomyosin complex
Authors: Tuchweber B. , Vonk R. J. , Yousef I. M.
Published: December 2009

Studies on isolated liver cell plasma membranes enriched in bile canaliculi from male rats treated with phalloidin show marked changes in the membrane polypeptides. Upon examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a protein component identical in mobility to the myosin standard was dramatically reduced, while that corresponding to actin was increased. It is suggested that a myosin-like protein may be necessary for the contractile function of the actin filaments in the liver cells. The observed modifications may be related to the decreased bile secretion and dilatation of bile canaliculi induced by phalloidin.
Purification and some characteristics of a Ca2+-activated proteolytic enzyme from beef cardiac muscle
Authors: Tolnai Susan
Published: December 2009

A calcium-dependent neutral proteinase was purified from beef cardiac muscle. The crude extract prepared from cardiac muscle was subjected to acid precipitation and salt fractionation and then further purified by column chromatography on Sepharose 6B, DE-52, and Sephadex G-200 columns in succession. The final preparation showed an 11 300 fold increase in specific activity of the Ca2+-activated enzyme. Average enzyme protein yield was 2.4 μg/g fresh tissue. The enzyme was maximally active at pH 7.6 in the presence of 4 mM calcium. Proportionality of enzyme activity in partially purified preparations was retained when activity was measured at 25 °C using casein as the substrate. The rate of proteolysis by the purified enzyme was linear for 60 min under similar assay conditions. Fractionation of muscle homogenates showed that 70 to 73% of the total enzyme activity was present in the 24 000 × g and 30 000 × g supernatants. The enzyme was labile in aqueous solutions and storage at 4 °C and −20 °C resulted in considerable loss of activity, unless glycerol (50% v/v) was added to the solution.
Formation of angiotensin II by tonin from partially purified human angiotensinogen
Authors: Grisé C. , Boucher R. , Thibault G. , Genest J.
Published: December 2009

The renin substrate (angiotensinogen) has been purified from outdated human blood bank plasma. A 100-fold purification was achieved by ammonium sulphate protein fractionation and four successive chromatographic procedures. We show that tonin, a serine protease enzyme found in submaxillary glands of the rat, cleaves the human plasma angiotensinogen, devoid of tonin inhibiting factor(s), at a pH optimum of 5–5.5. It generates a pressor substance that was identified as angiotensin (A) II. The rate of cleavage of the human angiotensinogen preparation by 1 nmol of renin or tonin was calculated to be 1320 nmol AI/h for renin and 26 nmol AII/h for tonin.
Interaction of selectively spin-labeled 70S ribosomes with tRNAPhe. An electron paramagnetic resonance study
Authors: Rodriguez Angel , Dugas Hermann
Published: December 2009

70S ribosomes from Escherichia coli, selectively spin labeled on the SH groups of proteins S18, S12, S21, S17, and L27, were used to study the formation of the tertiary complex ribosome–poly(U)–tRNAPhe. Most of these ribosomal proteins are located in the region of binding of tRNA. The electron paramagnetic resonance observable structural change suggests a loosening of the ribosome structure upon binding of the tRNA molecule.
Isolation of size homogeneous preparations of high molecular weight and low molecular weight fibrinogens
Authors: Phillips Hannah M.
Published: December 2009

Molecular sizes of fibrinogen (F) similar to FI, higher molecular weight form, and FII, lower molecular weight form, have been found by Lipinska and colleagues. A procedure has been developed to isolate for the first time each of the FI and FII forms of fibrinogen which are free of each other and of high molecular weight fibrin–fibrinogen complexes. This process involved removing the complexes by A-5 m chromatography. This chromatography also reduced a protein contaminant (X) and removed plasminogen. (NH4)2SO4 subfractionation at pH 5.9 was then done. A subtraction (16–18%) containing 90% FI and another (22–25% or 25–28%) containing 96% FII were obtained. Reprecipitation of the first 16–18% subfraction yielded a subfraction containing 97% FI. Sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis of FII revealed that it contains one intact Aα chain per (Bβ, γ)2. Clot opacity studies on FII suggested that the carboxyl terminal portion of the α chain of fibrin plays an important role in the lateral associations in fibrin polymerization.Also, the pattern of (NH4)2SO4 precipitation of the endogenous fibrin–fibrinogen complexes was studied. This revealed that the complexes precipitated mostly in the least soluble subfractions, but small amounts could be found in all subfractions. Examination of the complexes by SDS–polyacrylamide gel electrophoresis showed that most of the complexes could be dissociated to FI and FII. However, there were complexes which remained and these were found to be covalently cross-linked forms probably produced by factor XIII.
Tissue and species-specific effects of small molecular weight nuclear RNA's on transcription in isolated mammalian nuclei
Authors: Liu Wing-Cheong , Godbout Roseline , Jay Ernest , Yu Kenneth K.-Y. , Krause Margarida O.
Published: December 2009

Small molecular weight nuclear RNA's (SnRNA's) purified from the 0.35 M NaCl extract of chromatin from human and monkey tissues have been found to stimulate transcription of chromatin in isolated nuclei in a tissue- and species-specific manner. While SnRNA from normal human cells (WI38 fibroblasts and placenta) stimulates homologous transcription to some extent, it has a greater activity on transcription of heterologous tissue of the same species and no activity on heterologous tissue of a different species (monkey kidney cells). Likewise SnRNA from monkey cells stimulates transcription of homologous chromatin but has no effect on human cells. Fractionation of the RNA's in polyacrylamide gradient slab gels revealed that in all cases the active RNA was 160–175 nucleotides in length. Our results are compatible with the hypothesis that the active RNA's are involved in the determination and maintenance of tissue differentiation by recognizing promoter or regulator sequences in the DNA and act at the level of the nucleosome to induce tissue-specific genes.
Conformation of cross-linked aspartate transcarbamoylase
Authors: Chan William W.-C.
Published: December 2009

We have previously shown that aspartate transcarbamylase loses its substrate cooperativity after modification with a cross-linking reagent. Depending on the presence or absence of substrate analogues during cross-linking, the derivatives resemble the relaxed (R) or taut (T) state, respectively. In the present study, we attempt to characterize the conformation of these derivatives and the effects of ligands.The putative T-state derivative was similar to the native enzyme in its reactivity towards p-hydroxymercuribenzoate and in the increase of reactivity upon addition of succinate. However, unlike the native enzyme it was not activated by succinate at low substrate concentrations. On the other hand, the putative R-state derivative showed greatly enhanced reactivity which was not substantially increased by succinate. In the presence of urea, the native enzyme and the two cross-linked derivatives all resembled the R state. Thus at low substrate concentrations urea activated both the native enzyme and the T-state derivative. In contrast, the effect of urea on the R state derivative is mainly inhibitory.The above results show that the R state has been definitely stabilized whereas the T-state derivative retains some conformational flexibility. Our observations also indicate that the conformational change induced by succinate has two distinct components of which only one is allowed in the T-state derivative.
Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro
Authors: Ganz Peter R. , Kiss Gyorgy B. , Pearlman Ronald E.
Published: December 2009

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.
The glycosphingolipids of human plasma lipoproteins
Authors: Clarke J. T. R.
Published: December 2009

Human plasma contains low concentrations of four neutral glycosphingolipids (glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylcerarmide) and GM3 ganglioside which occur as part of the plasma lipoproteins, particularly low density lipoprotein (LDL, d 1.006–1.063 g∙mL−1) and to a lesser extent with high density lipoprotein (HDL, d 1.063–1.21 g∙mL−1). Plasma glucosylceramide appears to exchange freely between plasma lipoproteins and erythrocytes, and probably also between different lipoprotein fractions, in the circulation. Free exchange of other major neutral glycosphingolipids (GSLs) between lipoproteins and erythrocytes, or between lipoprotein fractions, does not normally occur. The GSL profile of each lipoprotein fraction is the same as the overall GSL composition of unfractionated plasma. In Fabry disease and Gaucher disease, GSL storage diseases, the excess glycolipid in plasma is distributed among the various lipoprotein fractions in the same relative proportions as in healthy individuals. In familial hypercholesterolemia, in which the levels of all plasma GSLs are elevated, the excess GSL is largely associated with the increased concentrations of LDL. In patients with hereditary hypolipoproteinemias, the levels of GSL in plasma are decreased less than those of other lipids. The relative excess of GSL in these patients is distributed among the remaining lipoprotein fractions. Excess GSL such as occurs in Fabry disease, does not appear to have a biologically significant effect on the physical stability of human LDL.
Factors affecting the 16α-hydroxylation of estrone 3-sulfate by guinea pig liver microsomes
Authors: Tsoulis C. J. , Hobkirk R.
Published: December 2009

The estrone 3-sulfate 16α-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O2 ratio of 0.64 caused 50% inhibition of activity. Since inhibition was also obtained in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate∙HCl it seems likely that the hydroxylase is a cytochrome P450 containing system. A fourfold increase in enzyme activity was brought about by 40 mM Mg2+ or Ca2+ while the same concentration of Mn2+ resulted in a twofold increase. Lesser increases were seen with Na+ or K+ and complete inhibition was obtained in the presence of Fe2+, Cu2+, or EDTA. When assayed in the presence of detergent concentrations sufficiently small to guard against cytochrome P450 destruction, it was found that Cutscum, Triton X-100, and Triton N-101 each caused greatest inhibition of enzyme activity. Lesser inhibition was apparent in the presence of Miranol H2M, cholate, or deoxycholate. The nonionic detergent, Brij 35, caused least inhibition of all and, when hepatic microsomes were treated with higher concentrations of Brij 35, about 80% of protein and over 95% cytochrome P450 were to be found in the 100 000 × g supernatant. Microsomal activity was more stable when stored at −20 °C in buffer containing glycerol, EDTA, and dithiothreitol than in buffer alone. Under best conditions only 10% of the hydroxylase activity was lost in one week.
Cell-free ammonia-oxidizing system of Nitrosomonas europaea: general conditions and properties
Authors: Suzuki Isamu , Kwok Siu-Chun , Dular Usha , Tsang D. C. Y.
Published: December 2009

Conditions essential for the preparation of active ammonia-oxidizing extracts of Nitrosomonas europaea were studied. The extracts were unstable during storage and required specific assay conditions for ammonia oxidation. Bovine serum albumin, spermine, or MgCl2 was required for ammonia oxidation and the concentration of phosphate determined the relative effectiveness of each activator, i.e., albumin being most effective in 0.1 M phosphate and spermine or MgCl2 at lower phosphate concentrations. Kinetic experiments showed a partial reduction of cytochrome c preceding the initiation of oxygen consumption due to ammonia oxidation.
Interaction of muscle glycolytic enzymes with thin filament proteins
Authors: Bronstein W. W. , Knull H. R.
Published: December 2009

Purified glycolytic enzymes were individually chromatographed through columns of Sepharose 4B containing a covalently bound F-actin–tropomyosin complex. Five of these enyzmes, aldolase, glyceraldehyde-phosphate dehydrogenase, lactate dehydrogenase, pyruvate kinase, and phosphoglycerate kinase were able to interact with the complex. Glucosephosphate isomerase, triosephosphate isomerase, phosphoglycerate phosphomutase, and enolase did not bind to the F-actin–tropomyosin matrix. One nonbinding enzyme, phosphoglycerate phosphomutase, was observed to interact with F-actin–tropomyosin if the column was preloaded with lactate dehydrogenase. Since at least four other glycolytic enzymes did not associate with actin directly, it is suggested that if a glycolytic enzyme complex exists, these nonadsorbing enzymes must interact with one or more of the enzymes which do bind to actin.
Metabolism of apolipoprotein A-I of chylomicrons in rats and humans
Authors: Vigne Jean-Louis , Havel Richard J.
Published: December 2009

After intravenous injection of a large amount of small chylomicrons into intact rats, the concentration of apolipoprotein (apo) A-I was increased by about 40% and remained at this elevated level as most of the chylomicron triglycerides were removed from plasma during the ensuing hour. This apo A-I rapidly left the chylomicrons and was transferred to lipoproteins of higher density. Such transfer of apo A-I did not occur when chylomicrons were incubated at comparably high concentrations with rat serum. In normolipidemic humans, the concentration of apo A-I and apo A-II, as well as phospholipids, increased in the light subfraction of high density lipoproteins (HDL2) 4 to 7 h after ingestion of a meal containing 1.5 g cream fat per kilogram body weight. The concentration of these components increased in the heavy subfraction of high density lipoproteins (HDL3) after 12 to 24 h. The concentration of apo E in plasma was unaffected by fat ingestion, but the concentration of this protein increased in lipoproteins of density less than 1.006 g∙mL−1 and fell in lipoproteins of higher density. It is concluded that apo A-I in rat chylomicrons is transferred quantitatively to HDL as chylomicron remnants are formed. Chylomicron apo A-I and apo A-II appear to be transferred similarly to HDL in humans, whereas apo E is transferred from HDL to chylomicrons after chylomicrons enter the blood.
Production of lipophorin in the fat body of adult Locusta migratoria: comparison with vitellogenin
Authors: Gellissen Gerd , Wyatt G. R.
Published: December 2009

The lipid-transport lipoprotein, lipophorin (Lp), an abundant plasma protein of insects, is synthesized in the fat body of Locusta migratoria as a polypeptide of relative mass (Mr) = 85 000. No posttranslational modification could be detected. Precipitation of this protein leads to formation of a product of Mr about 240 000, which fails to dissociate under treatment with sodium dodecyl sulfate and β-mercaptoethanol. In adult female locusts, synthesis and secretion of protein by the fat body increases three- to four-fold from emergence until day 7 of adult life, Lp representing 15–30% of the total. In the subsequent vitellogenic stage (days 7–14 or later), a further impressive increase in protein output up to five times the previtellogenic rate is observed, followed by a decrease to the previtellogenic level at the end of the cycle. This fluctuation is mainly due to production of the yolk precursor protein, vitellogenin (Vg), which makes up some 60% of the secreted protein at the peak of the cycle. Production of Lp does not follow these dramatic changes. Inactivation of the corpora allata, the source of juvenile hormone (JH), by treatment with precocene, prevents the synthesis of Vg in females, and diminishes the production of other proteins, including Lp, in both sexes. Normal secretion patterns can be restored by application of the JH analog, methoprene. It is concluded that, whereas the synthesis of Vg is dependent upon and intensely stimulated by JH, the synthesis of Lp is stimulated only to a minor extent, in common with other proteins.
Synaptosomal Na+,K+-ATPase as a membrane probe in studying the in vivo action of morphine
Authors: Wan-Kan Ophelia , Hosein E. A.
Published: December 2009

The activity of membrane-bound Na+,K+-ATPase was used as a metabolic probe to study the effects of morphine in vivo in rat brain synaptosomes. Arrhenius plots were generated to study an induced perturbation within the membrane. In acute studies 0.5-h postmorphine, the drug was without effect on the basal activity of the enzyme. With dopamine-stimulated Na+,K+-ATPase morphine decreased the apparent transition temperature and specific activity of the enzyme while there was a slight stimulation in its activation energy. An increase in these parameters was observed in samples taken from animals withdrawn from the drug for 48 h. These results strongly suggest the possible involvement of the membrane phospholipids as transducer which mediates the observed biphasic effect of the drug on enzyme activity.
Hepatic and intestinal contribution of two forms of apolipoprotein B to plasma lipoprotein fractions in the rat
Authors: Sparks Charles E. , Hnatiuk Oleh , Marsh Julian B.
Published: December 2009

The in vivo incorporation of labeled amino acids into two forms of apolipoprotein B of nascent hepatic, nascent intestinal, and plasma lipoproteins was studied. Using SDS–gel filtration column chromatography rat apolipoprotein B was separated into two proteins of higher (apo Bh) and of lower (apo B1) molecular size and the incorporation of label into each was measured. When livers isolated from fed rats were perfused with 3H-labeled amino acids, radioactivity was incorporated into both forms of apo B of the d < 1.060 fractions (very low (VLDL), intermediate (IDL), and low (LDL) density lipoproteins) with a labeling ratio of apo B1 to apo Bh of 0.8. When mesenteric lymph was collected from corn oil fed rats intraduodenally injected with 3H-labeled amino acids, radioactivity was mainly incorporated into apo B1 of chylomicrons and VLDL with apo B1 to apo Bh, labeling ratios of 14 and 44, respectively. Plasma was isolated 2 h after intraperitoneal injection of 3H-labeled amino acids into chow fed rats and lipoproteins were isolated by sequential density ultracentrifugation. The labeling ratio of apo B1 and apo Bh decreased from 4.2 in VLDL to 0.5 in LDL indicating a progressive enrichment of apo Bh in the LDL fraction. High density lipoproteins (HDL) contained less than 4% of the total labeled apo B and was enriched in apo B1. The results of this study indicate that the liver synthesizes both forms of apo B while the intestine synthesizes almost entirely apo B1. Since both apo B proteins are secreted primarily by the liver into VLDL, the results are consistent with preferential removal of apo B1 during triglyceride-rich lipoprotein catabolism and entry of hepatically derived apo Bh into LDL.
Plasma lipids, lipoproteins, and triglyceride turnover in eu- and hypo-thyroid rats and rats on a hypocaloric diet
Authors: Dory Ladislav , Krause Brian R. , Roheim Paul S.
Published: December 2009

Lipid and lipoprotein concentration, and triglyceride turnover were studied in control, thyroidectomized, and pair-fed control rats (pair-fed to match the food intake of the thyroidectomized rats). Thyroidectomy induced a significant increase in plasma cholesterol (and low density lipoprotein) concentrations and a decrease in plasma triglyceride (and very low density lipoprotein) concentrations. Changes in similar direction but of smaller magnitude were observed in the plasma of the pair-fed control rats. To further investigate triglyceride metabolism in these three groups of animals, triglyceride turnover was studied in fasted, unrestrained, and unanesthetized rats, following injection of [2-3H]glycerol. Peak incorporation of [2-3H]glycerol into plasma triglyceride occurred in all three groups of animals at 25 min after precursor administration, although the maximal incorporation was substantially lower in the thyroidectomized group than in either of the control groups. Thereafter, plasma triglyceride radioactivity decayed monoexponentially with a half-life of 24 ± 1 min for both normal and pair-fed control rats, compared with the half-life of 41 ± 3 min observed in the thyroidectomized rats. The calculated apparent fractional catabolic rates were thus 0.029 min−1 for both control groups and only 0.017 min−1 for the thyroidectomized animals. The apparent total catabolic rates of plasma triglyceride were 299 ± 11, 138 ± 11, and 48 ± 4 μg triglyceride∙min−1 for the normal controls, pair-fed controls, and thyroidectomized rats, respectively. These data further emphasize the importance of thyroid hormones in regulating plasma lipid and lipoprotein metabolism and, specifically, indicate that hypothyroidism results in a reduction of triglyceride secretion into, and the removal from, circulation. Furthermore, evidence was presented that the decreased caloric intake of the hypothyroid animals cannot, in itself, account for this observation.
Analysis of chromosome movement in crane fly spermatocytes by ultraviolet microbeam irradiation of individual chromosomal spindle fibres. I. General results
Authors: Sillers Peggy J. , Forer Arthur
Published: December 2009

Single chromosomal spindle fibres in anaphase Nephrotoma ferruginea (crane fly) spermatocytes were irradiated with monochromatic ultraviolet light focussed to a 4-μm spot by means of an ultraviolet microbeam apparatus. The movement of the half-bivalent associated with the irradiated spindle fibre was either unaffected or the half-bivalent stopped moving; i.e., the effect was all-or-none. When the half-bivalent associated with the irradiated spindle fibre did stop moving, the partner half-bivalent moving towards the opposite pole (i.e., the half-bivalent with which the first half-bivalent was previously paired) also stopped moving: all other half-bivalents moved normally. In over 90% of the 69 cases the movements of the two half-bivalents were only temporarily blocked; when movement resumed both half-bivalents resumed movement at the same time, after stoppage times ranging from 2 min to more than 15 min. In a few cases the half-bivalents never resumed poleward motion.When half-bivalents that had stopped movement finally resumed movement they often did not reach the poles; i.e., they "lagged" and remained separate from the other chromosomes. This result occurred only in spermatocytes of N. ferruginea. In spermatocytes of N. suturalis or N. abbreviata, on the other hand, the stopped half-bivalents did not lag but always reached the poles.Half-bivalent pairs that stopped moving in N. ferruginea spermatocytes did so for shorter times than did those previously reported (after irradiation of chromosomal spindle fibres) in N. suturalis spermatocytes. We suggest that the difference is due to our use of monochromatic ultraviolet light as opposed to the previous use of heterochromatic ultraviolet light. We assume that different wavelengths of monochromatic light produce different effects, that any given monochromatic irradiation produces only one effect (albeit different effects at different wavelengths), but that heterochromatic irradiations can produce multiple effects.Irradiation of the interzone (between separating half-bivalents) had no effect on the chromosome-to-pole movements of the half-bivalents. Therefore the stoppage of movement of half-bivalent pairs is specific for irradiation of chromosomal spindle fibres. On the other hand, irradiation of the interzone often blocked pole-to-pole elongation.
Regulation of ribonucleotide reduction by deoxyribonucleotides in intact Chinese hamster ovary cells
Authors: Hunting Darel , Henderson J. Frank
Published: December 2009

The ribonucleotide reductase model of Thelander and Reichard, which is based on studies of the purified enzyme, has been tested in cultured Chinese hamster ovary K-1 cells. Deoxyribo-nucleoside triphosphate concentrations were manipulated by the use of drugs or naturally occurring deoxyribonucleosides, and ribo- and deoxyribo-nucleoside triphosphate concentrations were measured. The results are consistent with the hypothesis that dTTP inhibits CDP reduction and stimulates GDP reduction, and that dGTP inhibits CDP reduction. However, the results are contrary to the hypothesis that dGTP inhibits UDP reduction and stimulates ADP reduction. and that dATP inhibits CDP reduction. It is concluded that the model of Thelander and Reichard applies only in part to intact CHO cells.
Renal metabolite concentrations and the activities of glutaminase and glutamate dehydrogenase during recovery from metabolic acidosis in the rat
Authors: Parry David M. , Hall Beatrice , Brosnan John T.
Published: December 2009

Metabolite content was determined in freeze-clamped kidneys to elucidate the rate-controlling steps which are responsible for the inhibition of renal ammoniagenesis that occurs when rats are allowed to recover from metabolic acidosis. After 1 day of recovery from acidosis there were increased renal contents of glutamate, glutamine, α-ketoglutarate, citrate, lactate, and malate. The calculated cytoplasmic concentration of oxaloacetate was also increased. The renal content of phosphoenolpyruvate, 3-phosphoglycerate, and ammonia decreased during recovery. No changes were observed in the renal content of the adenine nucleotides or of inorganic phosphate. The activities of phosphate-dependent glutaminase and glutamate dehydrogenase were elevated even after 7 days of recovery although the renal contents of glutamate and α-ketoglutarate had returned to control levels by this time. The changes in oxaloacetate and phosphoenolpyruvate are consistent with the fall in the activity of phosphoenolpyruvate carboxykinase observed by Parry and Brosnan. The increased levels of α-ketoglutarate and of glutamate are considered to be a consequence of a primary change in the activity of α-ketoglutarate dehydrogenase. These results are discussed in the light of the known effects of these metabolites on glutaminase activity and on glutamine entry into renal mitochondria.
The nucleotide sequence of phenylalanine tRNA and 5S RNA from Rhodospirillum rubrum
Authors: Newhouse N. , Nicoghosian Krikor , Cedergren R. J.
Published: December 2009

The complete nucleotide sequence of tRNAPhe and 5S RNA from the photosynthetic bacterium Rhodospirillum rubrum has been elucidated. A combination of in vitro and in vivo labelling techniques was used. The tRNAPhe sequence is 76 nucleotides long, 7 of which are modified. The primary structure is typically prokaryotic and is most similar to the tRNAPhe of Escherichia coli and Anacystis nidulans (14 differences of 76 positions). The 5S ribosomal RNA sequence is 120 nucleotides long and again typical of other prokaryotic 5S RNAs. The invariable GAAC sequence is found starting at position 45. When aligned with other prokaryotic 5S RNA sequences, a surprising amount of nucleotide substitution is noted in the prokaryotic loop region of the R. rubrum 5S RNA. However, nucleotide complementarity is maintained reinforcing the hypothesis that this loop is an important aspect of prokaryotic 5S RNA secondary structure. The 5S and tRNAPhe are the first complete RNA sequences available from the photosynthetic bacteria.
The effects of ergosterol and alcohols on germ-tube formation and chitin synthase in Candida albicans
Authors: Chiew Yoke Yin , Sullivan Patrick A. , Shepherd Maxwell G.
Published: December 2009

Ergosterol inhibited germ-tube formation and the membrane-bound enzyme chitin synthase in Candida albicans. The sterol solvents methanol and ethanol stimulated chitin synthase activity but inhibited germination. Methanol increased both the rate of protease activation of the chitin synthase proenzyme and the Vmax of the enzyme. The activation was not due to a change in the protease action on the proenzyme. The methanol activation was reversible and therefore the extraction of inhibitory components from the membrane was not causing the activation. Methanol had no effect on the N-acetylglucosamine activation sites. Analysis of the N-acetylglucosamine activation by an iteration programme gave Ka values of 0.3 mM for the high affinity sites and 20 mM for the low affinity sites. The degree of cooperativity with respect to the substrate uridine diphosphate N-acetylglucosamine was not changed by the presence of methanol and therefore the solvent did not affect the subunit–subunit interactions of the enzyme. Arrhenius plots of chitin synthase have discontinuities and methanol did not affect the transition temperature indicating that the structure of the membrane in the immediate vicinity of the enzyme was unchanged.
Surface potential and conductance induced in lipid bilayers by the negatively charged ionophore Br-X537A
Authors: Roy G. , Okada Y. , Laprade R.
Published: December 2009

The adsorption of Br-X537A and its effect on the surface potential of monoolein lipid bilayers was measured using the nonactin conductance as a probe to determine the surface charge density. Because of the pH dependence of this adsorption, it was concluded that not only the negatively charged molecules X− could induce a surface charge but also a dimer HX2− made from X− and the neutral molecule HX. Also an important bilayer conductance was induced by Br-X537A. From the Br-X537A concentration dependence of this conductance, the effect of pH, and the induced surface potential, it was found that two charged complexes are transported across the bilayer depending on pH. At pH ≥ 7 the conducting molecule is X− and at pH ≤ 5 the complex is H2X3−. A quantitive model is obtained to calculate both the induced surface potential and the conductance.