bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens
Authors: Mayr G W
Published: August 2015

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called ‘metal-dye detection’ has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.
Carbohydrate structures of the human-immunodeficiency-virus (HIV) recombinant envelope glycoprotein gp120 produced in Chinese-hamster ovary cells
Authors: Mizuochi T , Spellman M W , Larkin M , Solomon J , Basa L J , Feizi T
Published: August 2015

The present paper describes the structures of the N-linked oligosaccharides of the human-immunodeficiency-virus (HIV) envelope glycoprotein gp120 (cloned from the HTLV-III B isolate and expressed as a secreted fusion protein after transfection of Chinese-hamster ovary cells), which is known to bind with high affinity to human T4-lymphocytes. Oligosaccharides were released from peptide by hydrazinolysis, fractionated by paper electrophoresis, high-performance lectin-affinity chromatography and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. The glycoprotein was found to be unique in its diversity of oligosaccharide structures. These include high-mannose type and hybrid type, as well as four categories of complex-type chains: mono-, bi-, tri- and tetra-antennary, with or without N-acetyl-lactosamine repeats, and with or without a core-region fucose residue. Among the sialidase-treated oligosaccharides, no less than 29 structures were identified as follows: (formula; see text) where G is galactose, GN is N-acetylglucosamine, M is mannose, F is fucose, and ‘+/- ’ means that residues are present in a proportion of chains. The actual number of oligosaccharide structures is much greater, since before desialylation there was evidence that, among the hybrid and complex-type chains, all but 6% contained sialic acid at the C-3 position of terminal galactose residues, and partially sialylated forms of the bi- and multi-antennary chains were present. Detailed evidence for the proposed oligosaccharide sequences will be published as a supplementary paper [T. Mizuochi, M. W. Spellman, M. Larkin, J. Solomon, L. J. Basa & T. Feizi (1988) Biomed. Chromatogr., in the press].
The effect of chronic ethanol ingestion on protein metabolism in type-I- and type-II-fibre-rich skeletal muscles of the rat
Authors: Preedy V R , Peters T J
Published: August 2015

1. The effects of chronic ethanol feeding on muscles containing a predominance of either Type I (aerobic, slow-twitch) or Type II (anaerobic, fast-twitch) fibres were studied. Male Wistar rats, weighing approx. 90 g or 280 g, were pair-fed on a nutritionally complete liquid diet containing 36% of total energy as ethanol, or isovolumetric amounts of the same diet in which ethanol was replaced by isoenergetic glucose. After 6 weeks feeding, fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]-phenylalanine and muscles were analysed for protein, RNA and DNA. 2. Ethanol feeding decreased muscle weight, protein, RNA and DNA contents in both small and large rats. Type-II-fibre-rich muscles showed greater changes than did Type-I-fibre-rich muscles. Changes in protein paralleled decreases in DNA. 3. The capacity for protein synthesis (RNA/protein), fractional rates of protein synthesis and absolute rates of protein synthesis were decreased by ethanol feeding in both small and large rats. The amounts of protein synthesized relative to RNA and DNA were also decreased. Changes were less marked in Type-I than in Type-II-fibre-rich muscles. Loss of protein, RNA and DNA was greater in small rats, but protein synthesis was more markedly affected in large rats. 4. It was concluded that chronic ethanol feeding adversely affects protein metabolism in skeletal muscle. Fibre composition and animal size are also important factors in determining the pattern of response.
Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium
Authors: Danoff S K , Supattapone S , Snyder S H
Published: August 2015

Inositol 1,4,5-trisphosphate (InsP3) is a component of the phosphoinositide second-messenger system which mobilizes Ca2+ from intracellular stores. Recently, an InsP3 receptor binding protein from rat cerebellar membranes was solubilized and purified to homogeneity. The potent inhibition by Ca2+ of [3H]InsP3 binding to the InsP3 receptor in cellular membranes is not apparent in the purified receptor. The Ca2+-dependent inhibition of [3H]InsP3 binding in the crude homogenate (concn. giving 50% inhibition = 300 nM) can be restored by addition of solubilized cerebellar membranes to the purified receptor. In the present study, we further characterize the protein in solubilized membranes which confers Ca2+-sensitivity to the receptor, and which we term ‘calmedin’. Calmedin appears to be a neutral membrane protein with an estimated Mr of 300,000 by gel filtration in the presence of Triton X-100. Calmedin confers a Ca2+-sensitivity to InsP3 receptor binding, which can be completely reversed by 10 min incubation with EDTA and therefore does not represent Ca2+-dependent proteinase action. Calmedin effects on the purified InsP3 receptor depend on Ca2+ binding to the calmedin, although Ca2+ also binds directly to the InsP3 receptor. The regional distribution of calmedin differs from that of the InsP3 receptor in the brain, suggesting that it also mediates other Ca2+-dependent functions. Calmedin activity in peripheral tissues is much lower than in brain.
Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage
Authors: Sampaio L de O , Bayliss M T , Hardingham T E , Muir H
Published: August 2015

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.
Regulation of phenobarbital-inducible cytochrome P-450s in rat and mouse liver following dexamethasone administration and hypophysectomy
Authors: Meehan R R , Forrester L M , Stevenson K , Hastie N D , Buchmann A , Kunz H W , Wolf C R
Published: August 2015

Cytochrome P-450s are a superfamily of haem-containing proteins involved in the metabolism of foreign compounds, as well as a variety of endogenous molecules. The hepatic levels and function of this diverse group of enzymes are determined by both constitutive and xenobiotic regulators. To examine the role of constitutive factors in cytochrome P-450 regulation, the levels of three distinct groups of phenobarbital-inducible hepatic cytochrome P-450s were studied following dexamethasone-treatment or hypophysectomy. In the mouse, dexamethasone was a potent inducer of proteins within the PB1 (subfamily IIC), PB2c (family III) and PB3 (subfamily IIB) families. These findings were strikingly different from the effects in the rat where essentially no effect on PB3 expression and indeed suppression of proteins related to PB1 was observed. Determination of mRNA concentration indicated that the difference was at the level of transcription. These findings indicate that synthetic glucocorticoids have the potential to be potent phenobarbital-like inducing agents. In the mouse hypophysectomy, like dexamethasone, induced hepatic mRNA of P-450 from families P-450IIB, P-450IIC and P-450III. Again a species difference was observed as this treatment had essentially no effect in the rat. These data in the mouse indicate that factors produced in the pituitary can either affect the transcription rate of phenobarbital and dexamethasone-inducible P-450 genes or influence the stability of their mRNAs.
Ligninolytic enzymes of the white-rot fungus Phlebia radiata
Authors: Niku-Paavola M L , Karhunen E , Salola P , Raunio V
Published: August 2015

One oxidase (EC 1.10.3.2) and three lignin peroxidases (EC 1.11.1.-) were purified from the culture liquid of the white-rot fungus Phlebia radiata Fr. All the enzymes were glycoproteins. The oxidase had Mr 64,000 and the lignin peroxidases I, II and III had Mr values 42,000, 45,000 and 44,000 respectively. The lignin peroxidases were found to share common antigenic determinants: lignin peroxidases II and III were serologically indistinguishable and lignin peroxidase I was related but distinguishable. The oxidase did not share any immunological properties with the lignin peroxidases. Lignin peroxidases of Phlebia contain protoporphyrin IX as a prosthetic group. In the presence of H2O2 and an electron donor, veratryl alcohol, lignin peroxidases exhibit spectral shifts analogous to those of animal catalase (EC 1.11.1.6). Phlebia enzymes show optimal activity at pH 3-4.5 at 40 degrees C and are stable in the pH range 5-6. They modify Kraft lignin and phenolic compounds containing hydroxy and methoxy groups.
Chromosome-encoded β-lactamases of Citrobacter diversus. Interaction with β-iodopenicillanate and labelling of the active site
Authors: Amicosante G , Oratore A , Joris B , Galleni M , Frère J M , Van Beeumen J
Published: August 2015

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.
Immunological evidence for common antigenic sites in high-density lipoproteins from rainbow trout and man
Authors: Ayrault-Jarrier M , Burdin J , Fremont L , Gozzelino M T
Published: August 2015

The plasma high-density lipoproteins (HDL, rho 1.085-1.21 g/ml) of rainbow trout (Salmo gairdneri) have been shown to contain large amounts of apolipoprotein (apo) AI (Mr 28,000) and several other apolipoproteins of Mr less than 14,000 (apo C-like) and of Mr 37,000-38,000, 44,000-45,000 and 53,000-54,000. Comparison of apo AI from trout and human HDL shows them to be similar in Mr and to have some cross-immunoreactivity, whereas apo AII differs in Mr but also possesses common antigenic sites. It is suggested that the major apolipoproteins of fish and human HDL may fulfil similar roles in lipid transport.
A survey of the kinetic parameters of class C β-lactamases. Penicillins
Authors: Galleni M , Frère J M
Published: August 2015

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.
Methylthioadenosine toxicity and metabolism to methionine in mammalian cells
Authors: Christa L , Kersual J , Augé J , Pérignon J L
Published: August 2015

5′-Deoxy-5′-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5′-deoxy-5′-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.
Iodothyronine 5′-deiodinase activity and thyroid hormone content in brown adipose tissue during the breeding cycle of the rat
Authors: Viñas O , Giralt M , Obregón M J , Iglesias R , Villarroya F , Mampel T
Published: August 2015

Brown adipose tissue iodothyronine 5′-deiodinase activity is significantly lower in 17-day pregnant rats compared with virgin controls and remains low during late pregnancy and lactation. It fully recovers with abrupt weaning, but only partially with spontaneous weaning. Even though this profile of changes is remarkably in step with the known pattern of modifications in brown fat thermogenesis during the breeding cycle, the lowered iodothyronine 5′-deiodinase activity appearing between days 15 and 17 of pregnancy occurs earlier than the reduction in brown adipose tissue thermogenesis. Brown fat 3,3′,5-tri-iodothyronine content is also reduced in late pregnant, early and mid-lactating rats, most probably as a consequence of the lowered 5′-deiodination of thyroxine in situ. Acute insulin treatment increases brown fat iodothyronine 5′-deiodinase activity in virgin animals as well as in late-pregnant and lactating rats, despite the lowered basal enzyme activity levels in the latter groups. Thus an impaired response to insulin in brown fat does not appear to be a factor leading to the lowered iodothyronine 5′-deiodinase activity during late pregnancy and lactation.
Egasyn affects the processing of β-glucuronidase in mouse liver
Authors: Pfister K , Bosshard N , Zopfi M , Gitzelmann R
Published: August 2015

Three differently modified forms of beta-glucuronidase are known to exist: a microsomal enzyme form (M) existing in tissues where egasyn, a second microsomal protein, is present; and an acidic (La; complex-type oligosaccharide) and a basic (Lb; non-complex type oligosaccharide) lysosomal form which occur in all mouse tissues. Lb predominates in tissues containing microsomal beta-glucuronidase, La in those lacking it. In pulse-labelling experiments using mouse strain C57BL/6 liver containing egasyn (Eg+/Eg+) and microsomal enzyme, about half of the newly synthesized beta-glucuronidase was processed to the microsomal enzyme form, which was evidently further processed to Lb, and about half directly to La. In contrast, in liver of the congenic line C57BL/6.YBR Es-1b Eg0 that lacks egasyn (Eg0/Eg0) and microsomal enzyme, most of the labelled beta-glucuronidase was processed to La, and only a minor portion to Lb. Newly synthesized enzyme appeared first in microsomal, then in light and heavy lysosomal fractions of Eg+/Eg+ liver. In Eg0/Eg0 liver, no labelled enzyme was measurable in the microsomes, but it appeared rapidly in both types of lysosomes. Taken together these findings indicate that the microsomal enzyme form serves as a precursor of Lb, and that La is synthesized independently. The apparent half-life of La is only two-thirds that of Lb; this fact accounts for the reduced beta-glucuronidase activity in Eg0/Eg0 liver, which contains La as the predominant form.
Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol
Authors: Vrolix M , Raeymaekers L , Wuytack F , Hofmann F , Casteels R
Published: August 2015

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.
Purification and characterization of three forms of glutathione transferase from Proteus mirabilis
Authors: Di Ilio C , Aceto A , Piccolomini R , Allocati N , Faraone A , Cellini L , Ravagnan G , Federici G
Published: August 2015

Three forms of glutathione transferase (GST) with pI values of 6.0, 6.4 and 7.3 were isolated from Proteus mirabilis AF 2924 by glutathione-affinity chromatography followed by isoelectric focusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide-slab-gel electrophoresis, all forms proved to be composed of two subunits of identical (22,500) Mr. GST-6.0 and GST-6.4 together account for about 95% of the total activity, whereas GST-7.3 is present only in trace amounts. Extensive similarities have been found between GST-6.0 and GST-6.4. These include subunit molecular mass, amino acid composition, substrate specificities and immunological characteristics. GST-7.3 also cross-reacted (non-identity) with antisera raised against bacterial GST-6.0. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with the bacterial enzymes, indicating major structural differences between them and the mammalian GSTs. This conclusion is further supported by c.d. spectra.
A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions
Authors: Enrich C , Bachs O , Evans W H
Published: August 2015

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.
Suppression of snake-venom cardiotoxin-induced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases
Authors: Tzeng W F , Chen Y H
Published: August 2015

The incubation of 10(5) single neonatal rat cardiomyocytes with 1 microM-cardiotoxin in a bath medium, Tyrode solution in the presence of 1 mM-Ca2+, at 37 degrees C evoked the following chain of events. Firstly, there appeared a latent period of about 10 min during which the cells behaved normally. Neither lactate dehydrogenase nor ATP leaked from the cells. Cytosolic free Ca2+ increased considerably, as measured by the fluorescence intensity of fura-2-Ca2+ complex. At the same time a large portion of endogenous ATP was depleted. Secondly, after the latent period, the cell beating became irregular and eventually stopped. Thirdly, blebs appeared on the cell surface, leading to cell degeneration. If, before the appearance of blebs, the cells were washed with the bath medium exhaustively or incubated in the presence of the toxin antibody, cytosolic free Ca2+ and endogenous ATP returned to normal levels and cells resumed regular beating. Preincubation of the cells with 3.75 microM-flunarizine or 3.75 microM-diltiazem (both are Ca2+ antagonists), or 1.5 microM-fura-2 acetoxymethyl ester (a chelate for Ca2+), or 200 microM-leupeptin or 50 microM-antipain (both are proteinase inhibitors) considerably suppressed the toxin's ability to degenerate the cells. On the other hand, lysosomal proteinase inhibitor, autophage inhibitor, serine proteinase inhibitor, phospholipase inhibitor and calmodulin antagonist did not inhibit the toxin's activity. The results suggest that the toxin may act on the extracellular surface of intact cardiomyocytes to increase cytosolic free Ca2+. The subsequent cell degeneration may result from the activation of a Ca2+-dependent non-lysosomal proteolytic system.
Purification and characterization of the P-80 glycoprotein from human brain
Authors: Girgrah N , Cruz T F , Letarte M , Moscarello M A
Published: August 2015

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.
Pregnancy and pentobarbital anaesthesia modify hepatic synthesis of acylglycerol glycerol and glycogen from gluconeogenic precursors during fasting in rats
Authors: Zorzano A , Herrera E
Published: August 2015

1. Incorporation of gluconeogenic precursors into blood glucose and hepatic glycogen and acylglycerol glycerol was examined in 24 h-fasted virgin rats by using a flooding procedure for substrate administration. At 10 min after their intravenous injection, the conversion of alanine or glycerol into liver glycogen or acylglycerol glycerol was proportional to glucose synthesis. 2. In 24 h-fasted 21-day-pregnant rats, the incorporation of alanine and glycerol into hepatic acylglycerol glycerol was markedly enhanced compared with the control group. In addition, during fasting at late pregnancy, the proportion of substrates directed to acylglycerol glycerol as compared with the fraction incorporated into glucose was augmented. 3. In pentobarbital-treated fasted rats, the incorporation of both alanine and pyruvate into circulating glucose and into hepatic glycogen and acylglycerol glycerol was increased. Pentobarbital treatment increased the proportion of substrates incorporated into liver glycogen, compared with the fraction appearing in circulating glucose. These changes were concomitant with a marked accumulation of glycogen. 4. The data indicate that, during fasting, gluconeogenesis provides glucose as well as hepatic glycogen and acylglycerol glycerol, independently of whether the substrates enter gluconeogenesis at the level of pyruvate or dihydroxyacetone phosphate.
The role of a cathepsin D-like activity in the release of Galβ1-4GlcNAc α2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response
Authors: Lammers G , Jamieson J C
Published: August 2015

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose: N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.
Membrane proteins in senescent erythrocytes
Authors: Suzuki T , Dale G L
Published: August 2015

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.
Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method
Authors: Kijimoto-Ochiai S , Katagiri Y U , Hatae T , Okuyama H
Published: August 2015

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a ‘bisecting’ acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.
Biosynthesis of normal and low-molecular-mass complement component C1q by cultured human monocytes and macrophages
Authors: Hoekzema R , Brouwer M C , de Graeff-Meeder E R , van Helden H P T , Hack C E
Published: August 2015

High levels of low-molecular-mass complement component C1q (LMM-C1q), a haemolytically inactive form of C1q, are found in serum of individuals with inherited complete (functional) C1q deficiency and in serum of patients with systemic lupus erythematosus, whereas lower levels are present in normal serum [Hoekzema, Hannema, Swaak, Paardekooper & Hack (1985) J. Immunol. 135, 265-271]. To investigate whether LMM-C1q is a (by-)product of C1q synthesis or the result of degradation of C1q, cultures of blood monocytes and of alveolar macrophages, which secrete functional C1q, were studied. A considerable portion of C1q-like protein secreted by these cells was found to be LMM-C1q. In contrast with the C1q fragments that resulted from degradation of normal C1q during phagocytosis, culture-derived LMM-C1q appeared to be identical with LMM-C1q found in serum, as judged by sedimentation behaviour, subunit structure and recognition by poly- and mono-clonal antibodies raised against C1q. The presence of LMM-C1q in cytoplasmic organelles compatible with the Golgi apparatus and the inability to generate LMM-C1q by impeding hydroxylation and triple-helix formation of C1q further argues against degradation as its source. Monocyte cultures of homozygous probands from two families with complete functional C1q deficiency reflected the abnormalities in serum, i.e. absence of functional C1q, but increased levels of LMM-C1q. By contrast, secretion of C1q and LMM-C1q by cells from healthy individuals was clearly co-ordinate, indicating that LMM-C1q in serum may provide a unique marker of C1q synthesis in vivo.
Evidence that the production of acetate in rat hepatocytes is a predominantly cytoplasmic process
Authors: Crabtree B , Souter M J , Anderson S E
Published: August 2015

By using [1-14C]butyrate, the fluxes of butyrate to acetate and fatty acids were measured in rat hepatocytes. Both fluxes were inhibited to a similar extent by (-)-hydroxycitrate, with no significant effect on butyrate uptake. These results indicate that acetate formation takes place in the cytoplasm, presumably via ATP-stimulated acetyl-CoA hydrolase. Since acetate formation occurred despite a net uptake of acetate, the results are also consistent with the operation of a substrate cycle between acetate and acetyl-CoA, recently proposed by other workers, and suggest that this cycle is cytoplasmic.
The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII
Authors: Leyte A , Verbeet M P , Brodniewicz-Proba T , Van Mourik J A , Mertens K
Published: August 2015

The interaction between human Factor VIII and immobilized multimeric von Willebrand Factor (vWF) was characterized. Equilibrium binding studies indicated the presence of multiple classes of Factor VIII-binding sites on vWF. The high-affinity binding (Kd = 2.1 x 10(-10) M) was restricted to only 1-2% of the vWF subunits. Competition studies with monoclonal antibodies with known epitopes demonstrated that the Factor VIII sequence Lys1673-Arg1689 is involved in the high-affinity interaction with vWF.
28 S ribosomal RNA in vertebrates. Locations of large-scale features revealed by electron microscopy in relation to other features of the sequences
Authors: Wakeman J A , Maden B E H
Published: August 2015

The 28 S rRNA from several vertebrate species, when examined by electron microscopy, is seen to contain regions of extensive secondary structure, as first reported for HeLa-cell 28 S rRNA by Wellauer & Dawid [(1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2827-2831]. Here we correlate the locations of these regions, determined from the electron-microscopic data, with the primary structure of 28 S rRNA from human, mouse and Xenopus laevis determined by sequence analysis of rDNA. The secondary-structure features observed by electron microscopy correspond closely to phylogenetically variable G + C-rich regions that largely comprise the eukaryotic expansion segments in these three species. In most if not all cases the features can be identified with long G + C-rich helices deduced from sequence data. Correlations are given between the locations of the secondary-structure features and several ‘landmark’ restriction sites in 28 S rDNA. By correlating the locations of the rRNA methyl groups reported elsewhere [Maden (1988) J. Mol. Biol. 201, 289-314] with the present findings it is concluded that the rRNA secondary-structure features revealed by electron microscopy are largely or wholly unmethylated.
Bovine serum conglutinin is a lectin which binds non-reducing terminal N-acetylglucosamine, mannose and fucose residues
Authors: Loveless R W , Feizi T , Childs R A , Mizuochi T , Stoll M S , Oldroyd R G , Lachmann P J
Published: August 2015

Carbohydrate recognition by bovine serum conglutinin has been investigated by inhibition and direct binding assays using glycoproteins and polysaccharides from Saccharomyces cerevisiae (baker's yeast), and neoglycolipids derived from N-acetylglucosamine oligomers, mannobiose and human milk oligosaccharides. The results clearly show that conglutinin is a lectin which binds terminal N-acetylglucosamine, mannose and fucose residues as found in chitobiose (GlcNAc beta 1-4GlcNAc), mannobiose (Man alpha 1-3Man) and lacto-N-fucopentaose II [Fuc alpha 1-4(Gal beta 1-3)GlcNAc beta 1-3Gal beta 1-4Glc] respectively.
Studies on the inhibition by α-amanitin of single-step addition reactions and productive RNA synthesis catalysed by wheat-germ RNA polymerase II
Authors: de Mercoyrol L , Job C , Job D
Published: August 2015

The rate of formation of a single phosphodiester bond with UTP substrate, U-A primer, poly[d(A-T)] template and wheat-germ RNA polymerase II is greatly depressed in the presence of alpha-amanitin. Half-maximal inhibition occurs at 0.04 microgram/ml, in close agreement with published values for inhibition of productive RNA synthesis with class II RNA polymerases from higher-plant species. However, a sizeable proportion of U-A-U synthesis is resistant to inhibition by excess alpha-amanitin. In the additional presence of ATP, i.e. under experimental conditions permitting RNA chain elongation, the synthesis of poly[r(A-U)] is arrested after the formation of the first phosphodiester bond. The results support the contention that the main enzymic process disrupted by alpha-amanitin is the translocation step of the transcription complex along the DNA template.
High-level expression of fully active yeast flavocytochrome b2 in Escherichia coli
Authors: Black M T , White S A , Reid G A , Chapman S K
Published: August 2015

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from the yeast Saccharomyces cerevisiae and three singly substituted mutant forms (F254, R349 and K376) have been expressed in the bacterium Escherichia coli. The enzyme expressed in E. coli contains the protohaem IX and flavin mononucleotide (FMN) prosthetic groups found in the enzyme isolated from yeast, has an electronic absorption spectrum identical with that of the yeast protein and an identical Mr value of 57,500 estimated by SDS/polyacrylamide-gel electrophoresis. N-Terminal amino-acid-sequence data indicate that the flavocytochrome b2 isolated from E. coli begins at position 6 (methionine) when compared with mature flavocytochrome b2 from yeast. The absence of the first five amino acid residues appears to have no effect on the enzyme-catalysed oxidation of L-lactate, since Km values for the yeast- and E. coli-expressed wild-type enzymes were identical within experimental error. The F254 mutant enzyme expressed in E. coli also showed kinetic parameters essentially the same as those found for the enzyme from yeast. The R349 and K376 mutant enzymes had no activity when expressed in either yeast or E. coli. The yield of flavocytochrome b2 from E. coli is estimated to be between 500- and 1000-fold more than from a similar wet weight of yeast (this high level of expression results in E. coli cells which are pink in colour). The increased yield has allowed us to verify the presence of FMN in the R349 mutant enzyme. The advantages of E. coli as an expression system for flavocytochrome b2 are discussed.
Nitroprusside differentially inhibits ADP-stimulated calcium influx and mobilization in human platelets
Authors: Morgan R O , Newby A C
Published: August 2015

1. The effect of nitroprusside on cGMP concn., cAMP concn., shape change, aggregation, intracellular free Ca2+ concn. (by quin-2 fluorescence) and Mn2+ entry (by quenching of quin-2) was investigated in human platelets incubated with 1 mM-Ca2+ or 1 mM-EGTA. 2. Nitroprusside (10 nM-10 microM) caused similar concentration-dependent increases in platelet cGMP concn. and was without effect on cAMP concn. in the presence of extracellular Ca2+ or EGTA. 3. In ADP (3-6 microM)-stimulated platelets, nitroprusside caused 50% inhibition of shape change at 0.4 microM (+Ca2+) or 1.3 microM (+EGTA), aggregation at 0.09 microM (+Ca2+) and of increased intracellular Ca2+ at 0.02 microM (+Ca2+) or 2.1 microM (+EGTA). Entry of 1 mM-Mn2+ (-Ca2+) was inhibited by 80% by 5 microM-nitroprusside. 4. In ionomycin (20-500 nM)-stimulated platelets, nitroprusside (10 nM-100 microM) did not inhibit shape change or intracellular-Ca2+-increase responses, and only partially inhibited aggregation. 5. In phorbol myristate acetate (10 nM)-stimulated platelets, neither shape change nor aggregation was inhibited by 5 microM-nitroprusside. 6. The data demonstrate that nitroprusside inhibits ADP-mediated Ca2+ influx more potently than Ca2+ mobilization. Nitroprusside appears not to influence Ca2+ efflux or sequestration and not to affect the sensitivity of the activation mechanism to intracellular Ca2+ concn. or activation of protein kinase C.
Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase
Authors: Murphy G , Ward R , Hembry R M , Reynolds J J , Kühn K , Tryggvason K
Published: August 2015

The metalloproteinase ‘gelatinase’ stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.
Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors
Authors: Eskild W , Kindberg G M , Smedsrød B , Blomhoff R , Norum K R , Berg T
Published: August 2015

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.