bluuebunny/crossref_metadata_2025 huggingface dataset:
The determination of calcium-binding sites of human erythrocyte membranes
Authors: Moore R. Blaine , Dryden E. E. , Kells D. I. C. , Manery J. F.
Published: December 2009

Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation. The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites. A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates. On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 × 104 M−1 and n = 30 nmol/mg protein; site 2, Ka = 3 × 103 M−1 and n = 200 nmol/mg protein; site 3, Ka = ~102 M−1 and n = ~200 nmol/mg protein. Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2. The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3). Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. By the method of partition centrifugation two classes of binding sites were identified by computer analysis. Their association constants and capacities are, respectively, 1.1 × 105 M−1 and 20 nmol/mg protein for site 1 and 4.4 × 103 M−1 and 200 nmol/mg protein for site 2. We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.
Control of glycoprotein synthesis. IX. A terminal Manα1-3Manβ1- sequence in the substrate is the minimum requirement for UDP-N-acetyl-D-glucosamine:α-D- mannoside (GlcNAc to Manα1-3) β2-N-acetylglucosaminyltransferase I
Authors: Vella George J. , Paulsen Hans , Schachter Harry
Published: December 2009

Twenty low molecular weight compounds were tested as substrates for UDP-GlcNAc:α-D-mannoside (GlcNAc to Manα1-3) β2-N-acetylglucosammyltransferase I (GlcNAc-transferase I) purified from bovine colostrum. This enzyme is at a key control point in the biosynthetic path leading to complex Asn-linked oligosaccharides. The highest activity was obtained with the substrate Manα1-3(R1α1-6)Manβ1-R2 where R1 was Manα1-3(Manα1-6)Man- (Km = 0.20 mM) and R2 was -4GlcNAcβ1-4GlcNAc-Asn. Somewhat less effective were substrates in which R1 was Man- (Km = 0.4–0.6 mM) and R2 was either -4GlcNAc or -4GlcNAcβ1-4(Fucα1-6)GlcNAc-Asn. Removal of the Manα1-6 arm (R1 = H-) or replacing R2 with an isopropyl group had no effect on Vmax but increased the Km about 10-fold, thereby leading to an 85% reduction in enzyme activity as measured under standard conditions. An 85% reduction in activity was also observed if R2 was replaced with N-acetylglucosaminitol. Enzyme activity was reduced 33% if R1 was Galβ1-4GlcNAcβ1-2Man-. Any compounds lacking a Manα1-3- terminus or in which the β-linked Man had been replaced with an α-linked Man were totally inactive. It was concluded that a terminal Manα1-3Manβ1-sequence is a minimal structural requirement for a GlcNAc-transferase I substrate. The only effective substrate for partially purified UDP-GlcNAc:α-D-mannoside (GlcNAc to Manβ1-6) β2-N-acetylglucosaminyltransferase II (GlcNAc-transferase II) from bovine colostrum was R1GlcNAcβ1-2Manα1-3(Manα1-6)Manβ1-R2 where R1 = H-. The absence of a terminal GlcNAcβ1-2- residue or masking this residue by making R1 = Galβ1-4-, both prevented enzyme activity, indicating that GlcNAc-transferase I action must precede GlcNAc-transferase II action during biosynthesis of complex Asn-linked oligosaccharides.
Change in phenotype in long-term cultures of a clonal rat bone cell line: switch to the synthesis of α1 (I)-trimer collagen
Authors: Limeback Hardy , Otsuka Kichibee , Yao Kam-Ling , Aubin Jane E. , Sodek Jaro
Published: December 2009

A number of bone cell clones isolated from rat calvaria have been maintained in culture for more than 3 years. Several of these clones have undergone dramatic changes in phenotype. One of these clones, RGB 2.2, was observed originally to have a fibroblastic morphology in culture and to respond to parathyroid hormone (PTH), but not prostaglandin E2 (PGE2), with an increase in intracellular cAMP. Throughout several passages in early subcultures, these cells synthesized mostly type I collagen, with small amounts of type III and type V collagens. Whereas PTH had no detectable effect on collagen synthesis, PGE2 decreased the amount of total cell layer collagen, with the greatest effect on type III collagen, while increasing the proportion of type V collagen. Subsequent studies on these cells during 3 years in culture have indicated changes in their phenotype including a progressive change in morphology to a more cuboidal shape and a change in collagen synthesis, the cells producing large amounts of the "embryonic" collagen, α1(I) trimer. The reason(s) for the change in collagen expression is unknown, but may be the result of a change in which gene(s) is being expressed.
Control mechanisms in brown adipose tissue plasma membrane
Authors: Bégin-Heick N. , Heick H. M. C.
Published: December 2009

It is generally agreed that the site of heat production during nonshivering thermogenesis is the brown adipose tissue (BAT) and that the triggering event for heat production is the interaction of noradrenaline (NA) with its receptor on the plasma membrane. Following this initial event, several changes occur which result in increased rates of cAMP synthesis, redistribution of ions across the membrane, enhanced rates of lipolysis, and increased mitochondrial oxidation of substrates. BAT is also a target for the anabolic effect of insulin. Available evidence shows that insulin receptors are present on the BAT plasma membrane and that insulin can oppose the metabolic effects of catecholamine on BAT. We have studied more particularly the response of BAT adenylate cyclase to catecholamines in an animal model (the ob/ob mouse) which has a defective thermogenic response. The capacity of adenylate cyclase to be stimulated by catecholamines was significantly less in the tissue of obese mice than in lean controls. To produce a response equal to the half-maximal response in the lean mouse, a 10-fold increase in the NA concentration was required in the BAT of the obese mouse. These results are in harmony with those of others showing that the lipolytic response to catecholamines is abnormal in the BAT of the obese mouse. The adenylate cyclase activity can be altered by changes in the lipid composition of the diet and by manipulation of hormone levels. It is likely that the alteration in adenylate cyclase responsiveness is one of the contributing factors in the impaired thermogenesis and obesity in this animal.
The specific capsular polysaccharide of Streptococcus pneumoniae type 33F
Authors: Richards James C. , Perry Malcolm B. , Kniskern Peter J.
Published: December 2009

The specific capsular polysaccharide of Streptococcus pneumoniae type 33F (American type 70) is composed of D-galactose (5 parts), D-glucose (1 part), and O-acetyl (ca. 0.4 parts). Periodate oxidation, partial hydrolysis, and 1H and 13C nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight polymer of a repeating hexasaccharide unit having the structure:[Formula: see text]
Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II
Authors: Maguire Graham F. , Little J. Alick , Kakis Gary , Breckenridge W. Carl
Published: December 2009

Two previously unidentified apolipoproteins (apo) designated apo C-II-X and C-II-Y have been found in plasma of homozygotes and obligate heterozygotes of a family with apo C-II deficiency. Because the plasmas of homozygotes do not activate lipoprotein lipase, apo C-II-X and C-II-Y are apparently nonfunctional. These apolipoproteins have isoelectric focusing points of 5.15 and 5.54, respectively, compared with 4.88 and 4.74 for the known isomorphs, C-II-1 and C-II-2, respectively. They have approximately similar molecular weights to apo C-II-1 and C-II-2 on two-dimensional sodium dodecyl sulphate – glycerol – polyacrylamide slab gel electrophoresis. They do not form insoluble antigen–antibody complexes with antibodies to apo C-II in single antibody immunodiffusion or electroimmunoassay systems. However, using a double-antibody technique in which immunoblotting is coupled with polyacrylamide isoelectric focusing slab gel electrophoresis, apo C-II-1, C-II-2, C-II-X, and C-II-Y have similar reactivity with antibodies to apo C-II. In this family the presence of apo C-II-X and C-II-Y discriminates obligate heterozygotes from normal subjects.
Rapid qualitative changes in mRNA populations in cultured human lymphocytes: comparison of the effects of cycloheximide and concanavalin A
Authors: Forsdyke Donald R.
Published: December 2009

To examine the hypothesis that the stimulation of cultured lymphocytes by lectins involves the inactivation of a protein repressor of putative "activation genes," the effects of a protein synthesis inhibitor (cycloheximide) and a lectin (concanavalin A) were compared. Qualitative changes in mRNA populations were assessed by translating RNA prepared from cycloheximide- or lectin-treated cultures in a rabbit reticulocyte lysate. [35S]Methionine-labelled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Cycloheximide increased the radioactive labelling of cultured lymphocytes with the RNA precursor [3H]uridine, as previously reported. This was observed during the first 3 h of culture; thereafter, cycloheximide was inhibitory. The period of increased labelling with [3H]uridine coincided with a period of great increase in mRNA corresponding to an acidic protein of a relative mass of approximately 55 000. This mRNA was not detected in RNA prepared from control cultures, but was one of the most abundant mRNA species detected in RNA prepared from cycloheximide-treated cultures. Increases in certain less abundant mRNA species were also noted. However, the mRNAs were not observed in RNA prepared from lectin-treated cultures. If an increase in these mRNAs is important for lymphocyte activation, then the increase must be to an extent not detected by our current methods.
Effects of heparin fractions of different affinities to antithrombin III and thrombin on the inactivation of thrombin and factor Xa by antithrombin III
Authors: Cerskus Andrew L. , Birchall Kathy J. , Ofosu Frederick A. , Hirsh Jack , Blajchman Morris A.
Published: December 2009

To investigate the relative contribution of heparin-binding thrombin and antithrombin III to the enhancement of the rate of inactivation of thrombin by antithrombin III, standard heparin was fractionated on matrix-linked thrombin and (or) antithrombin III. There was a good correlation between heparin affinity for antithrombin III and its ability to enhance the inactivation of thrombin and factor Xa. In addition, there was a good correlation between affinity of heparin for thrombin and its catalytic activity on the inactivation of thrombin by antithrombin III. Thus fractions with high affinity to thrombin had similar rate-enhancing activity for thrombin inactivation to that of fractions with high affinity to antithrombin III. Fractions with high affinity to both proteins were more potent than fractions with high affinity to either protein alone. No significant differences in mean molecular weight were observed among the various heparin fractions. A heparin fraction with very low affinity to thrombin and high affinity to antithrombin III was prepared by repeated fractionation of a low molecular weight heparin on the two affinity columns. This fraction had very weak rate-enhancing activity for the inactivation of thrombin by antithrombin III, but retained substantial activity for the inactivation of factor Xa. The results of these studies support the concept that, for both standard and low molecular weight heparin, the enhancement of the inactivation of thrombin by antithrombin III requires the interaction of the heparin with both thrombin and antithrombin III.
Interaction of high and low density lipoproteins on glycosaminoglycan secretion by human vascular smooth muscle cells and fibroblasts
Authors: Wosu Leonard , McCormick Suzanne , Kalant Norman
Published: December 2009

Low density lipoprotein (LDL) increased secretion of glycosaminoglycans (GAG) and the cell cholesterol content of proliferating fibroblasts and smooth muscle cells in culture; with increasing cell density the GAG effect decreased, but the cholesterol effect did not. High density lipoprotein (HDL, d > 1.063) decreased GAG secretion by slowly proliferating cells; when cells were actively proliferating, HDL alone did not affect GAG secretion, but it inhibited the increase caused by LDL. Thus HDL appeared to influence GAG secretion by two separate mechanisms, an inhibition which was overcome by rapid proliferation and an anti-LDL effect. HDL2 (d = 1.063–1.100) partially reproduced the latter effect. In addition, HDL, HDL2, and HDL3 increased cell cholesterol; the ability of LDL to increase cholesterol was correspondingly reduced in the presence of HDL and its subfractions, suggesting that they act by common mechanisms.
Effects of different salts on the plasma membrane ATPase and on proton transport in yeast
Authors: Ahlers Jan
Published: December 2009

The effect of various inorganic and organic salts on the plasma membrane ATPase from the yeast Saccharomyces cerevisiae and on its proton transport were studied. We observed up to 460% stimulation of ATPase activity depending on the type of salt. The anion had a major effect and the monovalent cation a minor effect. These activations were the consequence of an increase in the affinity of the ATPase for activating Mg2+ ions and of an enhancement of Vmax. The mechanism of the reaction between enzyme, substrate, and Mg2+ ions was not altered by these salts. Similary, several salts stimulated the proton transport through the plasma membranes of Saccharomyces cerevisiae as evidenced by fluorescence quench of the dye 9-amino-6-chloro-2-methoxyacridine in plasma membrane vesicles. It is proposed that this effect is due to a stimulation of ATPase activity and in addition to charge compensation by membrane permeating anions.
Catalase: an old enzyme with a new role?
Authors: Percy Maire E.
Published: December 2009

Although animal catalase has been studied for decades, its physiological role has remained perplexing. It has two enzymatic functions, not only catalyzing the breakdown of H2O2 into O2 and H2O, but also in the presence of low concentrations of H2O2 catalyzing the oxidation of electron donors such as ethanol or phenols. In this article, I have summarized some well-known properties of the enzyme and have also described several recently discovered features. Of particular interest is the finding that, although catalase has been regarded as an intracellular enzyme, there is published evidence for its association with the plasma membrane of the erythrocyte. Moreover, recent work from my laboratory indicates that in vitro at alkaline pH in the presence of Mg2+, the biologically active diphenols (β-3,4-dihydroxyphenylalanine and the β-adrenergic agonists isoproterenol, norepinephrine, and epinephrine) appear to function as electron donor substrates for human erythrocyte catalase and inhibit the production of O2 from H2O2 at micromolar concentrations. The β-adrenergic antagonist propranolol inhibits O2 production much less effectively and appears to competitively inhibit the reaction of catalase with epinephrine. These observations suggest an analogy between catalase and the β-adrenergic hormone receptor and raise many questions of interest to basic science, health, and disease.
Membrane mythology: technical versus conceptual developments in the progress of research
Authors: Rothstein A.
Published: December 2009

The historical development of membrane research can be divided into three periods: "Early Membraneology" (approximately 1850–1940), "Classical Membraneology" (1940–1970), and "Modern Membraneology" (1970 to date). The "Early Period" is characterized by imaginative conceptual leaps based on technically simple experiments. Each of the latter periods is characterized by substantial increases in activity as measured by numbers of membrane-oriented investigators and of published papers. The transition into each new period is based on technical rather than conceptual breakthroughs. It is concluded that, in membrane research, technical advances tend to precede conceptual advances and that new technologies allow new directions in experimentation, leading to new levels of understanding.
Cell surface structural implications of some experiments with isolated glycolipids and glycoproteins
Authors: Grant Chris W. M.
Published: December 2009

A considerable amount of information has now accumulated which relates to the structure and behaviour of components of the eucaryote outer surface. It is thus possible to begin to derive a fairly detailed picture of this region of the cell membrane. This is particularly true for the carbohydrate chains of glycolipids and glycoproteins, about which there is good agreement. Such chains occupy totally hydrophilic environments. They possess considerable mobility relative to the macrostructures to which they are attached, yet they are constrained by rather severe potential energy barriers to oscillate about preferred conformations. Various polypeptides make up the other major outer surface feature. A point made here is that there is good reason to expect the membrane surface to be densely covered by protein, of which a substantial component will be material adsorbed from the surrounding environment. These features are illustrated in a scale drawing which emphasizes the potential for receptor modulation by interactions within the glycocalyx.
Association of the nucleocapsid protein N of vesicular stomatitis virus with phospholipids vesicles containing the matrix protein M
Authors: Capone John , Ghosh Hara P.
Published: December 2009

The matrix protein M and the nucleocapsid protein N were isolated from vesicular stomatitis virus and reconstituted into artificial phospholipid vesicles. While the M protein could be reconstituted into phospholipid vesicles, the N protein had no affinity for lipid vesicles. The N protein could, however, associate with phospholipid vesicles in the presence of M protein. Identical results were also obtained when an in vitro system synthesizing M and N proteins was used for reconstitution. The results suggest that M protein is involved in virus maturation by interacting with the viral envelope and the N protein of the nucleoprotein core.
The ATPase complex of Escherichia coli
Authors: Bragg Philip D.
Published: December 2009

The ATPase (ATP synthase) complex of Escherichia coli is composed of an extrinsic membrane protein (ECF1), which contains the active site for ATP formation and hydrolysis, and is attached to ECF0, a transmembrane protein through which protons move to or from the active site on ECF1. ECF1 is composed of five subunits (α–ε) with a stoichiometry of α3β3γδε. The stoichiometry of the three subunits (a–c) of ECF0 is probably a1b2c10–15. In addition to 3 mol tightly bound adenine nucleotide/mol ECF1, three other "exchangeable" nucleotide binding sites can be detected. These sites are still present in the α and β subunit defective ECF1 of uncA401 and uncD412 mutants, although some changes in the tightness of binding are evident. The active sites of ECF1 require normal a and p subunits and may be present at αβ subunit interfaces. Hydrolysis of ATP requires cooperative interactions between α and β subunits. At low concentrations of ATP, in the absence of added divalent cations, hydrolysis of this substrate can occur at a single site without release of the product. This is consistent with alternating or sequential site mechanisms for ATP hydrolysis or synthesis. Predictions of secondary and tertiary structures from the known primary amino acid sequences of polypeptides a, b, and c have led to the following conclusions. Polypeptide a forms six or seven transmembrane a helices. The amino-terminal sequence of polypeptide b spans the membrane, but most of the protein is exposed on the cytoplasmic surface of the membrane where it can be cleaved by proteases in vitro. Polypeptide c consists of two nonpolar membrane-spanning α helices linked by a polar segment at the cytoplasmic surface of the membrane. This loop region interacts with ECF1 or is close to the ECF1-binding site. This is shown by competition between ECF1 and antibody for binding to polypeptide c. Chemical modification of arginyl residues in the loop region of polypeptide c inhibits ECF1 binding. Protease cleavage of polypeptide b affects, but does not abolish, binding of ECF1 to ECF0. Presumably, polypeptide b interacts with ECF1 also. The individual roles of the ECF0 polypeptides in proton translocation are not clear. Mutants in any of the three polypeptides may be defective in proton translocation. However, mutant and chemical modification studies support a role for the polypeptide c oligomer in the transmembrane proton pathway.
Separation of nitrobenzylthioinosine from high affinity binding sites on human erythrocytes by gel chromatography
Authors: Paterson Alan R. P. , Almeida Anthony F. , Dahlig-Harley Ewa , Miller Jon P. , Cass Carol E.
Published: December 2009

The potent inhibitor of nucleoside transport, 6-[(4-nitrobenzyl)mercapto]-9-β-D-ribofuranosylpurine (NBMPR), binds reversibly, but with high affinity (KD 0.3–1.4 nM) to plasma membrane sites on human erythrocytes, and occupancy of those sites by NBMPR correlates with inhibition of transport. An earlier study from this laboratory showed that, upon photoactivation of site-bound [3H]NBMPR on erythrocyte membranes by exposure to UV light, isotopic ligand molecules became covalently linked to membrane polypeptides, which migrated as a discrete band on gel electrophoretograms, thereby identifying nucleoside transport polypeptides. The present study showed that erythrocytes on which the high affinity sites were occupied by reversibly bound [3H]NBMPR were freed of the latter by slow passage at 37 °C through 9 × 300 mm columns of Sephadex G-200 gel. Photoactivation of site-bound [3H]NBMPR on the intact cells caused apparent covalent attachment of the ligand because cells so treated retained the isotopic label during passage through the gel columns. Apparent covalent binding to erythrocytes also resulted from photoactivation of site-bound [3H]nitrobenzylmercaptopurine arabinoside.
Secretagogues cause a preferential discharge of large size granules in rat pancreas
Authors: Beaudoin Adrien R. , Grondin Gilles , Filion Mario , Lord André
Published: December 2009

We have compared the diameters of zymogen granules in the exocrine pancreas of fasted or stimulated rats. The average granule diameter was 0.73 μm ± 0.18 SD (sample size, 1460) in fasted animals and significantly reduced to 0.68 μm ± 0.17 SD (sample size, 860) after 120 min and two intraperitoneal injections of urecholine. Reduction of granule size was attributable to the preferential discharge of large size granules. The range between first and third quartiles of the distribution curve was identical in the two groups at 0.24 μm. There was no significant increase in the proportion of granules smaller than 0.40 μm after urecholine stimulation. A third group of animals was "hyperstimulated" by an infusion of a mixture of caerulein, secretin, and urecholine. After 210 min, the average granule diameter was reduced to 0.43 μm ± 0.14 SD (sample size, 786). The range between first and third quartiles of the distribution curve was 0.16 μm. In this group, 43.5% of the granules was smaller than 0.40 μm. Granule size reduction was accompanied by the appearance of numerous pleiomorphic condensing vacuoles. The present results strongly support the views that secretagogues cause the preferential release of large size granules and favor the formation of small size granules.
Séparation des virotoxines du champignon Amanita virosa et étude comparative de leur interaction sur l'actine in vitro
Authors: Turcotte Andrée , Gicquaud Claude , Gendreau Marthe , St-Pierre Serge
Published: December 2009

Cyclic peptides have been extracted with methanol from Amanita virosa and separated by preparative HPLC. Six peptides were obtained: phalloidin and five new peptides recently identified by Faulstich as virotoxins. We have compared the interaction of these six peptides with actin in vitro. They increased the rate of polymerization of actin and protected F-actin against several denaturating agents: proteases, heat, chaotropic ions, cytochalasin B, and DNAse I. The five virotoxins have therefore the same biological properties as phalloidin. However, the differential spectra of interaction between actin and the five virotoxins are different than the differential spectra between actin and phalloidin, thus it appears that the molecular interaction of actin with virotoxins is different than with phalloidin. The five virotoxins have the same activity. Although these virotoxins have different functional groups on amino acids 1 and 7, it is concluded that these two amino acids are of minor importance in the interaction of these peptides with actin.
Signal resistance of a soluble protein to enzymic proteolysis. An unorthodox approach to the isolation and purification of germin, a rare growth-related protein
Authors: Grzelczak Z. F. , Lane B. G.
Published: December 2009

Onset of growth in germinating wheat embryos is marked by the conspicuous synthesis of germin, a soluble homopentameric protein. Germin is unusually stable in reducing environments containing sodium dodecyl sulfate, but the polymeric form is converted to a protomer (ca. 26 kdaltons) by brief heat treatment. In respect to these physical properties, germin is similar to nucleoplasmin, the putative nucleosome-assembly factor in Xenopus oocytes. To expand the comparison, we treated germin with gastric pepsin in the expectation that pepsin-catalyzed hydrolysis of germin might generate a series of fragments of the kind derived by pepsin digestion of nucleoplasmin. To our surprise, germin was refractory under conditions used to degrade nucleoplasmin. Further study has shown that germin exhibits a measure of stability toward the action of broad-specificity proteases which is unprecedented for a soluble protein. In this report, we document the remarkable resistance of this growth-related protein to enzymic proteolysis and project how this property may make it possible to isolate and purify an otherwise intractably rare, but "interesting" protein.
Enzymatic sulfation of steroids. XIX. Cortisol sulfotransferase activity, glucocorticoid sulfotransferases, and tyrosine aminotransferase induction in chicken, gerbil, and and hamster liver
Authors: Singer Sanford S. , Galaska Edward G. , Feeser Theresa A. , Benak Robert L. , Ansel Anne Z. , Moloney Anne
Published: December 2009

Radioisotopic, pH 6.8 assays were designed to measure hepatic cortisol sulfation in chickens, gerbils, and hamsters of both sexes. Enzyme levels with 40 μM cortisol were similar in males of all three species and due mostly to low Km enzymes with 10–30 μM cortisol Km's. Maximum enzyme activity in male chickens required 40 μM Cortisol. In the other species, the much higher maximum enzyme activity observed required 500 μM cortisol owing to sulfotransferases with Km's for the hormone near 300 μM. Coenzyme 3′-phosphoadenosine-5′-phosphosulfate requirements also varied between species. Sex differences of the enzyme levels were found only in hamsters. There, males possessed only 24–33% of the enzyme levels found in females. Cortisol 21-sulfate was the reaction product in all of the species. Sexual dimorphism in hamsters appeared to be due to repressive effects of androgens. pH optima of enzyme activities in the three species ranged from pH 6 to 7. Routine use of pH 6.8 assays allowed representative interspecies comparisons. DEAE-Sephadex fractionation of cytosol showed that chicken liver contained mostly two enzymes with different pH optima that catalyzed cortisol sulfation. These differed from the enzymes that catalyzed dehydroepiandrosterone and estradiol sulfation. In the gerbil four enzymes with similar pH optima catalyzed cortisol sulfation. The second of these to elute from DEAE-Sephadex columns was the low Km form. In hamsters most glucocorticoid sulfotransferase activity appeared to be due to one enzyme. The molecular weights of the low Km gerbil enzyme and the main hamster enzyme were 98 300 ± 6100 and 105 000 ± 8100. Hamsters and gerbils responded to injection of cortisol by hepatic tyrosine aminotransferase induction.
Isolation of dissimilar components of the 8.5S nonactivated uterine progestin receptor
Authors: Tai Ping-Kaung Ku , Faber Lee E.
Published: December 2009

Sucrose gradient analysis of the binding of our monoclonal antibody (secreted by cell line KN 382/ECl) to [17 α-methyl-3H] R5020-labeled rabbit uterine progestin receptors revealed that the antibody bound only to the 8.5S form (Kd = 0.86 nM) and not to the 7S and smaller complexes. 125I-labeled antibody, on the other hand, bound to both the 8.5S complex and a component of dissociated receptor. Calculation of the relative mass (Mr) of the 125I-labeled immunoglobulin G1 (lgG1) – protein complex indicated the addition of a 60 000 Mr peptide. Electrophoretic analysis of immunoaffinity purified receptor substantiated this by revealing two protein bands [Formula: see text]. Sequential washing of adsorbed receptor was accompanied by dissociation of the bound steroid and the Mr 92 000 peptide. The [Formula: see text] peptide could only be completely eluted from the immunoadsorbent under denaturing conditions. Autofluorography of receptor complexes covalently bound with [17α-methyl-3H]R5020 revealed two bands, one with a [Formula: see text] and the second with a [Formula: see text]. Upon immunoprecipitation both peptides precipitated with the [Formula: see text] and [Formula: see text] peptides. Gel electrophoresis demonstrated that the [Formula: see text] peptide and the [Formula: see text] did not comigrate. We have concluded that the nondissociated 8.5S rabbit uterine progestin receptor complex contains [Formula: see text] and [Formula: see text] nonsteroid-binding components, in addition to the [Formula: see text] and [Formula: see text] steroid binders and that the antibody preferentially recognizes the [Formula: see text] nonsteroid-binding peptide.
Rabbit liver phosphorylase a phosphatase: regulation by glucose and caffeine
Authors: Monanu Michael O. , Madsen Neil B.
Published: December 2009

The modulation of three molecular forms of liver phosphorylase a phosphatases, the "catalytic" subunit phosphatase (35 000 relative mass (Mr)) and phosphatases 2A1 and 2A2, by glucose and caffeine and some physiologically important compounds were studied, using 32P-labelled phosphorylase a obtained from rabbit liver as substrate. Glucose and caffeine showed independent and additive activations. The caffeine effect was seen at micromolar to millimolar concentrations and glucose caused activation even at concentrations below the normal blood glucose level. The nucleotides ATP and AMP, at their presumed physiological concentrations in the liver, were strongly inhibitory. Inhibition by these nucleotides and other inhibitors tested showed varied responses to the presence of the activators glucose and caffeine, depending on the phosphatase form. Thus, significant relief of ATP inhibition was afforded by glucose and caffeine acting independently for the 35 000 Mr phosphatase, whereas relief of inhibition for phosphatases 2A1 and2A2 required a combination of glucose, caffeine, andMg2+. The Km of the liver 35 000 Mr phosphatase was about 50 μM for the liver substrate as compared with 4 μM for the muscle substrate. The Km of phosphatase 2A2 was about 16 μM and for phosphatase 2A1 it was about 20 μM, using liver substrate in the absence of any stimulators. Mg2+ inhibited the 35 000 Mr phosphatase, but became stimulatory for phosphatase 2A2 and was an almost obligatory requirement for phosphatase 2A1. The additive glucose and caffeine effects observed with respect to the inhibitory activities seen in this study are consistent with the view that glucose alone may not be the sole physiological regulator of glucose homeostasis through its action on phosphorylase a in the liver.
Kinetics of the hydrolysis of cellobiose catalysed by β-glucosidase
Authors: Hsuanyu Yuchiong , Laidler Keith J.
Published: December 2009

The hydrolysis of cellobiose catalysed by β-glucosidase has been investigated by experimental techniques which allow the course of reaction to be followed continuously. They involve assaying the product glucose by the use of ATP, hexokinase, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADP); the latter is converted into its reduced form NADPH which absorbs strongly at 340 nm. Rates were measured at nine pH values varying from 5 to 6.9, at substrate concentrations varying from 0.2 to 3.2 mM, and at temperatures varying from 10 to 37 °C. The pH dependence revealed pK values of 4.9 and 6.5 in the free enzyme at 24 °C, and these are little changed on complex formation. The rates measured over a range of temperature, as interpreted by Arrhenius plots, revealed an inflexion at 23 °C, found consistently under all conditions. The results are analyzed in terms of the mechanism[Formula: see text]It was found possible to obtain, for the four elementary reactions, activation energies and entropies of activation which explain the inflexion at 23 °C and the Arrhenius behavior above and below that temperature. Profiles are constructed showing the variations of entropy and enthalpy during the course of an individual reaction.
Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine
Authors: Proulx P. , Aubry H. , Brglez I. , Williamson D. G.
Published: December 2009

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid – bile salt micelles, such that the lecithin – bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid – bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained. The presence of Ca2+ stimulated the incorporation substantially. The results as a whole indicate that fatty acid uptake into brush border membrane is very responsive to a variety of conditions which could prevail in the gut during the absorption process.
Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells
Authors: Manganaro F. , Kuksis A.
Published: December 2009

We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol 3-phosphate.
Characterization by nuclear magnetic resonance of the concanavalin A binding oligosaccharide on the βb chain of placental β-hexosaminidase B: lectin binding to the separated polypeptide chains of hexosaminidases A and B
Authors: O'dowd Brian F. , Mahuran Don , Cumming Dale , Lowden J. Alexander
Published: December 2009

The type and distribution of the oligosaccharides on each polypeptide of human placental β-hexosaminidases A (α(βaβb)) and B (2((βaβb)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the βa chain contains negligible carbohydrate, the βb chain contains both the high-mannose and a complex type oligosaccharide, and the α chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the βb polypeptide of β-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.
Trypsin digestion of bovine cardiac troponin C in the presence and absence of calcium
Authors: McCubbin William D. , Kay Cyril M.
Published: December 2009

The rate of tryptic digestion of cardiac troponin C (cTNC) has been shown to be dependent on Ca2+ as was noted earlier for skeletal TNC (sTNC). Two representative peptides have been characterized on the basis of amino acid composition and partial amino terminal sequence analysis. Circular dichroism and fluorescence studies monitored their response to the presence of Ca2+. Their ability to form complexes with the ATPase inhibitory subunit of cardiac troponin (cTNI) was determined by urea –polyacrylamide gel electrophoresis and fluorescence experiments. The ability of these peptides to substitute for whole cTNC in restoring the ATPase activity of a partially inhibited synthetic actomyosin system was also explored. The N-terminal peptide 1–88 already contains a large amount of ordered structure, which indicates that the α-helices flanking binding site II of cTNC exist independently of Ca2+. Consequently this peptide shows limited increase in structure in the presence of Ca2+. It binds to cTNI independently of the presence of Ca2+ and could substitute for whole cTNC by relaxing the inhibitory effect of cTNI. The C-terminal peptide 103–158 has a low amount of secondary structure in the absence of Ca2+ but this increases dramatically in the presence of this cation. This peptide could only form a stable complex with cTNI in the presence of Ca2+ and was unable to release the inhibitory effect of cTNI.
The use of monoclonal antibodies to probe human apolipoprotein B structure and function
Authors: Milne Ross W. , Marcel Yves L.
Published: December 2009

Apolipoprotein (apo) B plays an important role in plasma lipid transport and in the maintenance of cholesterol homeostasis. Attempts to determine the structure of apo B have been hampered by technical obstacles resulting from its chemical and physical properties. Recently monoclonal antibodies (Mabs) against human apo B have been used as probes to study apo B structure and heterogeneity. Certain Mabs are capable of blocking binding of low density lipoprotein (LDL) apo B to the cell surface LDL receptor, which presumably reflects the proximity of their antigenic determinants to the receptor recognition domain. The distribution of antigenic determinants recognized by Mabs has been studied on the hepatic (apo B-100) and intestinal (apo B-48) forms of apo B and on fragments generated by limited proteolysis of apo B. Some Mabs are specific for apo B-100, whereas others cross-react with apo B-48. Apo B-100 specific Mabs coupled to Sepharose have been used to isolate separately apo-B-containing lipoproteins of intestinal and hepatic origin and their respective lipid and apolipoprotein compositions have been determined. Using the separated fractions it has been shown that apo B-100, but not apo B-48, can react with the LDL receptor. Most Mabs failed to react with apo B which had been delipidated and resolubilized, but in some cases immunoreactivity could be recovered if the solubilized apo B were reincorporated into lipid vesicles. These experiments showed that different determinants had different lipid requirements for their expression. Within an individual there is immunochemical heterogeneity in apo-B-containing lipoproteins in the expression of apo B antigenic determinants which can be detected by Mabs. Intersubject differences in reactivity of lipoprotein subfractions with Mabs have also been observed and in some cases appear to represent genetic polymorphism of apo B.
Triacylglycerol as a precursor in phospholipid biosynthesis in cultured neuroblastoma cells: studies with labeled glucose, fatty acid, and triacylglycerol
Authors: Cook H. W. , Spence M. W.
Published: December 2009

Neuroblastoma cells rapidly incorporate exogenous fatty acids into cellular triacylglycerol and relationships between triacylglycerol and phospholipid biosynthesis have been indicated by the relative time course of labeling of these lipids. To evaluate this further, neuroblastoma cells were labeled using potential precursors of phospholipid including radiolabeled triacyglycerol, glycerol, glucose, and fatty acid. With [2-3H]glycerol or a mixture of [2-3H]glycerol trioleate and glycerol tri[1-14C]oleate, phospholipids were labeled at very low levels (< 0.1 and < 0.5%, respectively). With [6-3H]glucose, labeling of lipids (0.5–3.5%) was greatest in medium containing 19 mM fructose, whereas labeling with [1-14C]18:2(n-6) was similar in media containing either 19 mM fructose or 25 mM glucose. Labeling of the glycerol moiety of triacylglycerol with [6-3H]glucose increased with 40–200 μM 18:2(n-6) present and occurred predominantly in 2 h. Some [6-3H]glucose label was in fatty acyl chains (chiefly 16:0) of triacylglycerol by 16 h, but was unaffected by exogenous 18:2(n-6). Triacylglycerol was the only lipid to increase in mass (threefold with 200 μM 18:2(n-6)). During the chase of cells pulsed with [6-3H]glucose, label in triacylglycerol declined within 0.5 h, whereas in phospholipid it increased transiently up to 2 h and then declined. Changes were inversely proportional to 18:2(n-6) levels in the chase medium and labeled acyl chains moved in parallel with the glycerol moiety. Thus, a major portion of acyl chain transfer from triacylglycerol was accompanied by glycerol. Triacylglycerol appears to serve as an expandable intracellular reservoir during an influx of acyl chains and subsequent incorporation of those acyl chains into phospholipid seems to involve some de novo phospholipid synthesis. As phospholipid mass does not change appreciably, such synthesis must be accompanied by equally rapid catabolism and turnover of membrane phospholipid.
1H nuclear magnetic resonance studies of ytterbium-substituted porcine intestinal calcium-binding protein
Authors: Shelling Judith G. , Hofmann Theo , Sykes Brian D.
Published: December 2009

The addition of ytterbium to calcium-saturated porcine intestinal calcium-binding protein resulted in the appearance of broad lanthanide-shifted resonances well outside the normally observed region of the 1H nuclear magnetic resonance spectrum of the calcium form of the protein. Variation of the salt concentration and temperature have led us to conclude that aggregation and chemical exchange do not contribute to the line widths of these resonances. Assuming that the line broadening of these lanthanide-shifted resonances arises from the contribution of the susceptibility line-broadening mechanism for protein residues proximal to the bound Yb3+ ion, we have calculated Yb3+–proton distances for nuclei in the metal-binding site. These lanthanide-shifted resonances provide very sensitive probes of the structure of the protein in solution.
Ca2+ -specific fluorescence changes in N-dansylaminoethyl-labelled oncomodulin
Authors: Mutus Bulent , Flohr Elizabeth J. , MacManus John P.
Published: December 2009

Rat oncomodulin (isoelectric point (pI) = 3.9) and homologous carp parvalbumin (pI = 3.95) was labelled at Cys-18 which is located in the AB loop, with the fluorescent reagent N-dansylaziridine. The fluorescence spectrum of the labelled parvalbumin was insensitive to both Mg2+ and Ca2+ binding. In contrast the dansyl fluorescence emission spectrum of labelled oncomodulin was quenched ~20% by Ca2+ in the presence of physiological concentrations of Mg2+. Approximately 90% of the Ca2+-induced fluorescence quenching was complete at a Ca2+/dansyl-S-aminoethyloncomodulin ratio of ~1. The labelled oncomodulin was insensitive to Mg2+ binding. The present study indicates that oncomodulin, unlike parvalbumin, undergoes a large Ca2+-specific perturbation that can be felt over the entire molecule. In addition this Ca2+-specific perturbation appears to be complete upon occupation of only one metal-binding site.
Interaction of histone H1 with oligonucleosomes in vitro provides a convenient method for isolating mononucleosomes
Authors: Boulikas Teni
Published: December 2009

The interaction of histone H1 with oligonucleosomes was studied in 0.15 M NaCl. Exogenous calf thymus H1 was added to a mixture of oligonucleosomes, soluble in 0.15 M NaCl, and the partially precipitated chromatin was removed by centrifugation. A strong preference of H1 for the longer nucleosomes in the mixture was observed causing their precipitation and leaving the shorter oligonucleosomes preferentially soluble. Histone H1 could even discriminate in its binding between mono- and di-nucleosomes, binding to dinucleosomes and leaving the mononucleosomes preferentially soluble. On this basis a convenient method was developed for the rapid isolation of mononucleosomes. The fidelity of H1 reconstitution to the oligonucleosomes under these conditions was suggested employing zero-length carbodiimide cross-linking and radioiodinated H1. This fraction of mononucleosomes displays significantly higher levels in the more hydrophilic H3.2 and H3.3, as well as H2A.2 and H2A.3 variant histone forms, compared with bulk chromatin. The, presumably, lower affinity of histone H1 for this subfraction of mononucleosomes is discussed.