bluuebunny/crossref_metadata_2025 huggingface dataset:
Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into S-phase
Authors: Panet R , Snyder D , Atlan H
Published: August 2015

In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.
The ornithine requirement of urea synthesis. Formation of ornithine from glutamine in hepatocytes
Authors: Lund P , Wiggins D
Published: August 2015

In hepatocytes, urea synthesis from glutamine is independent of added ornithine, even when rates are high after stimulation of glutamine metabolism by dibutyryl cyclic AMP, phenylephrine or vasopressin. Incubation with glutamine increases tissue [ornithine]. The increases parallel those of [N-acetylglutamate] under different conditions. The ornithine requirement of urea synthesis increases with increasing supply of ammonia. A function of the unique, highly regulated, glutaminase of liver may be to regulate ornithine synthesis.
Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins
Authors: Wolf C R , Seilman S , Oesch F , Mayer R T , Burke M D
Published: August 2015

The properties of five structurally related forms of cytochrome P-450 (PB1a, PB1b, PB2a, PB2b and PB2d) isolated from rats treated with phenobarbital have been compared with two forms isolated previously now termed ‘PB1c’ and ‘PB2c’ These enzymes were characterized by their marginal inducibility by phenobarbital and are clearly distinguishable from the major phenobarbital-inducible proteins. PB1a and PB1b differed in Mr (52,700 and 52,900), absorption spectra and papain-proteolysis fragments. However, they had identical N-terminal sequences. PB2a, PB2b and PB2d had apparent Mr values of 52,900, 52,900 and 50,800. PB2a and PB2b had different N-terminal sequences and, after digestion with papain, gave different papain-proteolysis fragments. The N-terminal sequence of PB2b was similar to, but not identical with, that of pregnenolone-16 alpha-carbonitrile-inducible P-450 species, and PB2b was the protein most closely related to PB2c. The extent of immunocross-reactivity among the forms was stronger within, than between, the PB1 and PB2 groups. Even structurally similar forms were functionally diverse, exhibiting large differences in metabolic specificity in the dealkylation of a series of alkoxyresorufins.
Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions
Authors: Thornton D J , Nieduszynski I A , Oates K , Sheehan J K
Published: August 2015

Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a ‘beaded’ structure and a contiguous filamentous ‘tail’ at one end and others which appeared as round ‘blobs’, some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the ‘blob-like’ appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.
Specificity of protein phosphatases in the dephosphorylation of protein kinase C
Authors: Parker P J , Goris J , Merlevede W
Published: August 2015

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.
Metabolism of apolipoprotein E-containing human plasma lipoproteins by rat and human cells in culture
Authors: Ranganathan S , Matsuura H , Yamamoto M , Kottke B A
Published: August 2015

Cultured preadipocytes from rat epididymal fat pads were able to bind, internalize, and degrade human plasma very-low-density lipoproteins (VLDL) more efficiently than low-density lipoproteins (LDL). VLDL, but not LDL, activated acyl-CoA: cholesterol acyltransferase (ACAT) and increased cholesterol accumulation in these cells. However, trypsin-treated VLDL (T-VLDL) lost the capacity to bind, activate ACAT, and increase cholesterol accumulation. After the treatment of VLDL with trypsin, SDS/polyacrylamide-gel electrophoresis and immunoblotting showed that apolipoprotein E (apo E) was completely degraded, whereas apolipoprotein CII (apo C-II) was preserved. ApoE complexed with dimyristoyl phosphatidylcholine (DMPC) was able to complete with VLDL for binding to the cells. Although T-VLDL did not bind to the preadipocytes, these cells accumulate triacylglycerols from T-VLDL, presumably after lipolysis, as efficiently as from native VLDL. Rat smooth muscle cells and skin fibroblasts also bind and metabolize human VLDL better than LDL. However, human skin fibroblasts and omental preadipocytes metabolized LDL better than VLDL. These studies indicate that rat tissues can recognize and metabolize apoE-containing human plasma VLDL although they cannot recognize human LDL.
Purification, N-terminal amino acid sequence and properties of hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli
Authors: Hart G J , Abell C , Battersby A R
Published: August 2015

Hydroxymethylbilane synthase (porphobilinogen deaminase) was purified to apparent homogeneity from Escherichia coli. The enzyme is a monomer of Mr approx. 40,000. The Km for porphobilinogen and relative Vmax. values have been obtained at various pH values over the range 6.2-8.8, enabling pK values for ionizable groups important for activity to be determined. The N-terminal amino acid sequence is presented.
Copper transport from Cu(I)-thionein into apo-caeruloplasmin mediated by activated leucocytes
Authors: Schechinger T , Hartmann H J , Weser U
Published: August 2015

A study on the transfer of copper from Cu-thionein into apo-caeruloplasmin, using Cu-thionein that was previously oxidised by activated leucocytes, was performed. Cu(I)-thiolate oxidation was conveniently monitored by the progressive decline of the specific Cotton bands between 400 and 300 nm. The characteristic e.p.r. properties and NN-dimethyl-p-phenylenediamine oxidase activity indicated a successful formation of caeruloplasmin. Taking into account the simultaneous occurrence of leucocytes, apo-caeruloplasmin and Cu-thionein in blood plasma, such an interaction would favour a possible metabolic link between either copper protein.
Species variants of cathepsin L and their immunological identification
Authors: Mason R W
Published: August 2015

Cathepsin L variants purified from sheep and ox liver are shown to have similar catalytic properties to those from rat, rabbit and man with regard to activity against the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide and inhibition by benzyloxycarbonyl-Phe-Phe-diazomethane, thus identifying cathepsin L in these species for the first time. All five variants of cathepsin L are shown to be immunologically related by their interaction with antibodies raised to the human enzyme. Sheep liver was found to yield more enzyme than any other species, suggesting that this tissue is a good source of cathepsin L. Cathepsin S, a closely related enzyme, could not be detected in livers of any of these species.
Collagen defects in lethal perinatal osteogenesis imperfecta
Authors: Bateman J F , Chan D , Mascara T , Rogers J G , Cole W G
Published: August 2015

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.
Computer simulations of the kinetics of irreversible enzyme inhibition by an unstable inhibitor
Authors: Topham C M
Published: August 2015

Computer simulations of the irreversible inhibition of an enzyme by an unstable inhibitor are presented. Data obtained at the end point of reaction are shown to conform poorly in many situations with relationships derived from integrated rate equations by setting t = infinity, and the implications concerning the experimental use of this method to determine kinetic constants describing inactivation are considered. The alternative approach of conducting experiments under conditions of inhibitor excess over enzyme is further discussed, and a graphical procedure is suggested for the description of time courses of reaction of enzyme with unstable inhibitor when an enzyme-inhibitor adsorptive complex is involved.
Stimulation of choline release from NG108-15 cells by 12-O-tetradecanoylphorbol 13-acetate
Authors: Liscovitch M , Blusztajn J K , Freese A , Wurtman R J
Published: August 2015

The effects of the potent tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on phosphatidylcholine (PtdCho) metabolism were investigated in the neuroblastoma X glioma hybrid cell line NG108-15. TPA (100 nM) stimulated by 150-200% the release into the medium of 3H radioactivity from cells that had been pre-labelled with [3H]choline. H.p.l.c. analysis of the medium revealed that TPA stimulated the release of only free [3H]choline (212 +/- 11% of control), without affecting such other labelled metabolites as [3H]phosphocholine and [3H]glycerophosphocholine. This effect was concentration-dependent, with a half-maximal effect obtained at 27.5 +/- 6.8 nM, and was observable as early as 5-10 min after exposure to TPA. The TPA-induced release of [3H]choline into the medium was accompanied by a small and variable decrease in cellular [3H]PtdCho (to 93 +/- 4% of control). However, the radioactivity associated with water-soluble cellular choline metabolites (mainly [3H]phosphocholine and [3H]glycerophosphocholine) remained unchanged. TPA also stimulated the release of [3H]choline derived from [3H]PtdCho that had been produced via the methylation pathway from [3H]methionine. These data suggest that phosphatidylcholine may serve as the source of free choline released from the cells in response to TPA. The possible enzymic mechanisms underlying this response are discussed.
Nuclear-envelope vesicles as a model system to study nucleocytoplasmic transport. Specific uptake of nuclear proteins
Authors: Riedel N , Fasold H
Published: August 2015

In the preceding paper [Riedel & Fasold (1987) Biochem. J. 241, 203-212] we have described a procedure for the preparation of nuclear-envelope vesicles (NE vesicles) from rat liver nuclei. These vesicles, which are largely free of components of the nuclear interior, were employed in an assay system in vitro to study protein translocation across the NE. We found that nuclear proteins such as histones, high-mobility-group proteins and acidic chromosomal proteins are specifically taken up and accumulated in the NE vesicles, whereas there is little or no affinity for non-nuclear proteins like immunoglobulin, myoglobin and cytochrome c. The kinetics of histone uptake into the NE vesicles are similar to those obtained for whole rat liver nuclei, and comparative studies with non-vesicular NEs prepared by deoxyribonuclease I-treatment (DNAase-NEs) indicate that the NE of the vesicles affects the uptake kinetics and increases the capacity for nuclear proteins. The uptake of histones into NE vesicles, but not the binding to DNAase-NEs, can be stimulated by GTP and GDP. Furthermore, we found that even very large molecules can be entrapped in the vesicles during their preparation. These results indicate that the NE vesicles might provide a useful system in vitro with which to investigate the structures and mechanisms involved in protein translocation across the NE.
The occurrence of N-methyl-d-aspartic acid in muscle extracts of the blood shell, Scapharca broughtonii
Authors: Sato M , Inoue F , Kanno N , Sato Y
Published: August 2015

A novel ninhydrin-positive compound, N-methyl-D-aspartic acid, was identified in the muscle extracts of the blood shell, Scapharca broughtonii. This compound is already known to have potent neuroexcitatory activity, inducing hypermotility and strong releasing action of serum luteinizing hormone in mammals. This may be, however, the first finding of N-methyl-D-aspartic acid in natural products.
Procollagenase activator produced by rabbit uterine cervical fibroblasts
Authors: Ishibashi M , Ito A , Sakyo K , Mori Y
Published: August 2015

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.
Physicochemical characterization of human intestinal lactase
Authors: Lau H K F
Published: August 2015

Human lactase was isolated from solubilized small-intestinal brush-border membranes by a combination of chromatography on concanavalin A-Sepharose, Bio-Gel 1.5m and chromatofocusing, with a yield of approx. 1% and a 750-fold purification. The enzyme appeared to be homogeneous on SDS/polyacrylamide-gel electrophoresis under both reduced and non-reduced conditions, with an apparent Mr of approx. 170,000. On gel filtration, however, it displayed an apparent Mr of approx. 380,000. The protein had a pI of 4.8, as judged by the chromatofocusing experiment, and had a lactase activity whose optimum is at pH 6.0. In addition to the beta-galactosidase activity, the protein also hydrolysed to various extents cellobiose, phlorizin, p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-glucoside, o-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-fucoside. Antisera had been raised against the purified enzyme in two rabbits. One of the antibody populations could inhibit the enzyme in a concentration-dependent manner. This antibody population was used to set up an antibody-bound Sepharose column for the use in an immunoaffinity purification of lactase from crude intestinal homogenate. A partially purified preparation of lactase could thus be obtained. The antibody population was also used to set up a radioimmunoassay for quantifying the enzyme. The competition assay could detect about 0.5 micrograms of lactase protein/ml.
β-d-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo
Authors: Sobue M , Habuchi H , Ito K , Yonekura H , Oguri K , Sakurai K , Kamohara S , Ueno Y , Noyori R , Suzuki S
Published: August 2015

A series of aryl and alkyl O-beta-D-xylosides and their analogues with S, NH or CH2 in the glycosidic linkage were prepared and examined for their ability to act as artificial chain initiators of chondroitin (dermatan) sulphate synthesis in embryonic chick cartilage, foetal rat skin and 6-week-old-rat aorta under conditions where normal protein-core synthesis was inhibited by cycloheximide. For all these tissues in culture, phenyl O-beta-D-xyloside and phenyl beta-D-thioxyloside were clearly more effective than the corresponding N-xyloside and homo-C-xyloside. Introduction of a carboxy group to the para position of their aglycone yielded derivatives with far lower initiator activity. In a concentration range lower than 0.1 mM, the effectiveness of alkyl beta-D-thioxyloside was greatly influenced by the carbon number (n) of the alkyl group and was at a maximum at n = 7 or 8 for the cartilage, at n = 5 for the skin and at n = 4 for the aorta. In the beta-xyloside-treated cartilages, the average length of newly formed chondroitin sulphate chains reflected the chain-initiator activity of added xyloside, i.e. the higher the initiator activity, the shorter the average chain length. In the skin and aorta, none of the drugs could relieve the inhibition of heparan sulphate synthesis caused by cycloheximide. Fertilized hens' eggs were each injected on day 9 with 9.2 mumol of beta-xyloside and the skeletal systems of embryos were examined a week later. The embryos treated with beta-xylosides of relatively high initiator activity showed a 30-40% decrease in the overall growth rate of skeletons, whereas those treated with beta-xylosides of low initiator activity showed little or no decrease in the growth rate. The results are consistent with the notion that the observed change in skeletal morphology results mainly, if not completely, from beta-xyloside-induced synthesis of core-protein-free chondroitin sulphate, and further suggest that a procedure employing a series of beta-xyloside homologues with various initiator activities will furnish an easily applied criterion on which to test the specificity of xyloside action on biological processes.
Purification of phosphatidylinositol kinase from bovine brain myelin
Authors: Saltiel A R , Fox J A , Sherline P , Sahyoun N , Cuatrecasas P
Published: August 2015

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.
Mechanisms of intracellular protein catabolism. Intracellular fate of microinjected polypeptides translated in vitro
Authors: Gaskell M J , Heinrich P C , Mayer R J
Published: August 2015

Erythrocyte-mediated microinjection was used to introduce [35S]polypeptides translated in vitro into 3T3-L1 cells. Such [35S]polypeptides are not degraded after loading into erythrocytes and are stable for the first 2 h after microinjection into growing 3T3-L1 cells. Similarly, little or no degradation of microinjected [35S]polypeptides is observed in either growing or confluent 3T3-L1 cells over a 70 h period. Microinjection of reticulocyte lysate alone does not affect the rate of degradation of long-lived endogenous protein. Reductively [3H]methylated lysate haemoglobin is degraded after microinjection by a cytosolic mechanism. Microinjected 125I-labelled bovine serum albumin is rapidly degraded by a cytosolic mechanism at the same rate in the absence or presence of reticulocyte lysate. The data do not support the notion that the observed lack of degradation of microinjected [35S]polypeptides translated in vitro is due to the presence of proteolytic inhibitors in reticulocyte lysates which can inhibit the degradation of microinjected or cellular proteins.
The synthesis of lysylfluoromethanes and their properties as inhibitors of trypsin, plasmin and cathepsin B
Authors: Angliker H , Wikstrom P , Rauber P , Shaw E
Published: August 2015

The synthesis of two lysylfluoromethanes is described by an extension of the synthesis method of Rauber, Angliker, Walker & Shaw [(1986) Biochem. J. 239, 633-640]. Ala-Phe-Lys-CH2F was found to be an active-centre-directed inhibitor of plasmin and trypsin, as is the corresponding chloromethane. However, the rate of covalent-bond formation is about an order of magnitude lower at 25 degrees C for the fluoro derivative. It was, in addition, an extremely effective inactivator of cathepsin B at pH 5.4 and 6.4. The chemical reactivity of fluoromethanes was compared with that of chloromethanes as alkylators of GSH. At pH 7.4 and 37 degrees C, a fluoromethane has 1/500th the reactivity of a chloromethane. A comparison of the rates of reaction of the fluoromethane with cathepsin B and with GSH at pH 6.4 revealed an enhancement of 10(8)-fold for the alkylation of the enzyme, ascribable largely to a proximity effect.
The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature
Authors: el Battari A , Luis J , Martin J M , Fantini J , Muller J M , Marvaldi J , Pichon J
Published: August 2015

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.
Effect of polyamine depletion on macromolecular synthesis of the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes
Authors: Assaraf Y G , Abu-Elheiga L , Spira D T , Desser H , Bachrach U
Published: August 2015

DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, prevented the increases in putrescine and spermidine, but not in spermine, in human erythrocytes infected with the malarial parasite Plasmodium falciparum. The addition of putrescine to these polyamine-depleted cultures restored the normal concentrations of spermidine, whereas that of putrescine even exceeded that of the control cultures. DFMO also inhibited the incorporation of radioactive amino acids into the proteins of parasitized erythrocytes. Electrophoresis on polyacrylamide gels revealed that the synthesis of some proteins was completely blocked by DFMO, but the synthesis of others was not affected. DFMO also caused a partial inhibition of RNA synthesis, and DNA synthesis was completely blocked in polyamine-depleted parasitized erythrocytes. It has been suggested that putrescine and/or spermidine are required for the synthesis of certain proteins in parasitized erythrocytes and that at least one of those proteins is related to the synthesis of DNA of the malarial parasite. It appears that polyamines regulate the schizogony process of P. falciparum.
Further characterization of the cold agglutinin from the snail Achatina fulica
Authors: Mitra D , Sarkar M , Allen A K
Published: August 2015

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.
Purification and photoaffinity labelling of a rat cytosolic binding protein specific for 3-methylcholanthrene
Authors: Arnold P S , Garner R C , Tierney B
Published: August 2015

Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.
Chemical taxonomy of the hinge-ligament proteins of bivalves according to their amino acid compositions
Authors: Kikuchi Y , Tamiya N
Published: August 2015

The proteins in the hinge ligaments of molluscan bivalves were subjected to chemotaxonomic studies according to their amino acid compositions. The hinge-ligament protein is a new class of structure proteins, and this is the first attempt to introduce chemical taxonomy into the systematics of bivalves. The hinge-ligament proteins from morphologically close species, namely mactra (superfamily Mactracea) or scallop (family Pectinidae) species, showed high intraspecific homology in their compositions. On the other hand, inconsistent results were obtained with two types of ligament proteins in pearl oyster species (genus Pinctada). The results of our chemotaxonomic analyses were sometimes in good agreement with the morphological classifications and sometimes inconsistent, implying a complicated phylogenetic relationship among the species.
Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor
Authors: Glatz Z , Kovár J , Macholán L , Pec P
Published: August 2015

Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.
Organization and sequence of the human α-lactalbumin gene
Authors: Hall L , Emery D C , Davies M S , Parker D , Craig R K
Published: August 2015

A recombinant bacteriophage containing the entire alpha-lactalbumin gene was isolated from a human genomic library constructed in bacteriophage lambda L47. Within this recombinant the 2.5 kb alpha-lactalbumin gene is flanked by about 5 kb of sequence on either side. The complete nucleotide sequence of the gene and its immediate flanking sequences were determined and compared with those of the rat alpha-lactalbumin gene. These studies showed that the size, organization and sequence of the exons have been highly conserved, whereas the introns have diverged considerably. In particular, the first intron of the human gene was found to contain an Alu repetitive sequence not present in the rat. A high degree of homology (67%) was also observed in the 5′ flanking regions, extending as far as 655 nucleotide residues upstream of the transcriptional initiation site. Comparison of the 5′ flanking sequences of these two alpha-lactalbumin genes with those of five casein genes has revealed the presence of a highly conserved region [consensus sequence: RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA], extending from position -140 to -110 in all seven sequences examined, suggesting a possible regulatory role in the hormonal control or tissue-specific expression of milk protein genes in the mammary gland.
Evidence that the slow conformation change controlling NADH release from the enzyme is rate-limiting during the oxidation of propionaldehyde by aldehyde dehydrogenase
Authors: Blackwell L F , Motion R L , MacGibbon A K H , Hardman M J , Buckley P D
Published: August 2015

The displacement of NADH from the aldehyde dehydrogenase X NADH complex by NAD+ was followed at pH 7.0, and the data were fitted by a non-linear least-squares iterative procedure. At pH 7.0 the decay constants for the dissociation of NADH from aldehyde dehydrogenase X NADH complexes (1.62 +/- 0.09 s-1 and 0.25 +/- 0.004 s-1) were similar to the values previously determined by MacGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462] at pH 7.6, and apparent differences between these values and those reported by Dickinson [(1985) Biochem. J. 225, 159-165] are resolved. Experiments at low concentrations of propionaldehyde show that isomerization of a binary E X NADH complex is part of the normal catalytic mechanism of the enzyme. Evidence is presented that the active-site concentration of aldehyde dehydrogenase is halved when enzyme is pre-diluted to low concentrations before addition of NAD+ and substrate. The consequences of this for the reported values of kcat. are discussed. A general mechanism for the aldehyde dehydrogenase-catalysed oxidation of propionaldehyde which accounts for the published kinetic data, at concentrations of aldehyde which bind only at the active site, is presented.
Formation, characterization and detection of a ternary complex between S protein, thrombin and antithrombin III in serum
Authors: Preissner K T , Zwicker L , Müller-Berghaus G
Published: August 2015

S protein, a plasma glycoprotein with Mr 78,000, has been shown to interfere with the heparin-catalysed inhibition of thrombin by antithrombin III. This interaction was further evaluated in the present study. Native human blood was replaced by either radiolabelled antithrombin III or radiolabelled prothrombin in the reaction mixture, which was incubated at 37 degrees C. At various time intervals the serum formed from the incubated blood was withdrawn and analysed by crossed immunoelectrophoresis against anti-(S protein) serum in the second dimension. Increasing quantities of radioactivity originating both from antithrombin III and from thrombin were precipitated in a cathodal shoulder to the S protein peak. This observation indicated the formation of a ternary S protein-thrombin-antithrombin III (STAT) complex in serum. This complex could also be observed by the same technique after incubation of purified thrombin in the presence of antithrombin III and S protein. Complex-formation was independent of the presence of heparin and did not require Ca2+ ions. Owing to the association of S protein with the thrombin-antithrombin III (TAT) complex, the STAT complex assembled in vitro exhibited a higher Mr than the TAT complex as judged by polyacrylamide-gradient-gel electrophoresis in the absence of SDS. Both the serum-originated STAT complex and the STAT complex assembled from purified components sedimented faster than the single components and showed comparable apparent sedimentation coefficients in the range 11-14 S, corresponding to a mean Mr of 350,000. The STAT complex could be detected in serum at a dilution of 1:3200 by a sensitive immuno-radiometric assay employing affinity-purified IgG against S protein. These results indicate that S protein, in addition to its role as a heparin-neutralizing factor, becomes incorporated into the nascent TAT complex or can bind to preformed TAT complex during the clotting process.
Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Characterization of haem and iron-sulphur centres by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy
Authors: Godfrey C , Coddington A , Greenwood C , Thomson A J , Gadsby P M A
Published: August 2015

The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.
Effects of lactation and removal of pups on the rate of triacyglycerol/fatty acid substrate cycling in white adipose tissue of the rat
Authors: Hansson P , Newsholme E A , Williamson D H
Published: August 2015

The rate of the triacylglycerol/fatty acid substrate cycle was measured in vivo in adipose tissue of virgin and lactating rats with pups removed. The rate decreased by 70% in adipose tissue of lactating rats and increased 9-fold on removal of the pups. Similar differences in cycling rate were seen in adipose tissue incubated in vitro in the presence of isoprenaline.
Effect of putrescine on the synthesis of S-adenosylmethionine decarboxylase
Authors: Kameji T , Pegg A E
Published: August 2015

The synthesis of S-adenosylmethionine (AdoMet) decarboxylase was studied by translating the rat prostate mRNA for this enzyme in a reticulocyte lysate. The protein was formed as a precursor of Mr 37,000, which was converted into the enzyme subunit of Mr 32,000 in the lysates. The presence of putrescine had no effect on the synthesis of the precursor of AdoMet decarboxylase, but accelerated its conversion into the enzyme subunit. Spermidine, spermine, decarboxylated AdoMet, AdoMet and methylglyoxal bis(guanylhydrazone) were not able to substitute for putrescine in this effect. These results indicate that, in addition to its direct activation of mammalian AdoMet decarboxylase, putrescine could increase the amount of the enzyme by increasing its production.
Subsecond and second changes in inositol polyphosphates in GH4C1 cells induced by thyrotropin-releasing hormone
Authors: Tashjian A H , Heslop J P , Berridge M J
Published: August 2015

It has been demonstrated previously that thyrotropin-releasing hormone (TRH) induces changes in inositol polyphosphates in the GH3 and GH4C1 strains of rat pituitary cells within 2.5-5.0 s. TRH also causes a rapid rise in cytosolic free calcium concentration ([Ca2+]i) in these cells which is due largely to redistribution of cellular calcium stores. Therefore, it has been concluded that TRH acts to release sequestered calcium in these cells via enhanced generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. If this conclusion were correct, TRH-enhanced accumulation of Ins(1,4,5)P3 should occur at least as rapidly as the increase in [Ca2+]i. We have shown previously that the rise in [Ca2+]i induced by TRH occurs within about 400 ms; thus, it was important to investigate the subsecond time-course of changes in inositol phosphates caused by TRH. Using a rapid mixing device, we have measured changes in inositol polyphosphates on a subsecond time scale in GH4C1 cells prelabelled with myo-[2-3H]inositol. Although TRH did alter inositol polyphosphate metabolism within 500 ms, the changes observed did not reveal a statistically significant increase in Ins(1,4,5)P3 within time intervals of less than 1000 ms. Thus, we have been unable to demonstrate that a TRH-induced rise in Ins(1,4,5)P3 precedes or occurs concomitantly with the rise in [Ca2+]i in GH4C1 cells. Although these results do not disprove the current view that Ins(1,4,5)P3 mediates the action of TRH on intracellular calcium redistribution, we conclude that caution should be exercised in this, and possibly other cell systems, in accepting the dogma that all of the rapid, agonist-induced redistributions of intracellular calcium are mediated by Ins(1,4,5)P3.
Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum)
Authors: Ramadoss C S , Shenoy B C , Borthakur A
Published: August 2015

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.
Neomycin cannot be used as a selective inhibitor of inositol phospholipid hydrolysis in intact or semi-permeabilized human platelets. Aminoglycosides activate semi-permeabilized platelets
Authors: Polascik T , Godfrey P P , Watson S P
Published: August 2015

High concentrations of neomycin (2-10 mM) inhibited aggregation, but not shape change, of intact platelets by collagen, ADP and the Ca2+ ionophore, A23187, the last two studies being carried out in the presence of the cyclo-oxygenase inhibitor indomethacin. In contrast, over the same range of concentrations neomycin inhibited both aggregation and shape change induced by thrombin. Under these conditions activation of platelets by collagen and by thrombin, but not by A23187 or by ADP, is believed to be dependent on the hydrolysis of membrane inositol phospholipids. These data therefore suggest that the inhibitory action of neomycin on intact platelets is not related to its previously reported inhibitory effect on phosphoinositide metabolism. The selective inhibition of thrombin-induced shape change indicates a second site of action of neomycin on intact platelets. On platelets rendered semi-permeable with saponin, neomycin and a second aminoglycoside antibiotic, streptomycin (each 0.06-2 mM), stimulated secretion and aggregation responses. These effects were inhibited by indomethacin and by EGTA. Activation of semi-permeabilized platelets by neomycin is associated with the formation of inositol phosphates and phosphatidic acid, indicating activation by phospholipase C. This effect is also inhibited by indomethacin, implying that it is secondary to the formation of prostaglandins and endoperoxides. These results are discussed in the context of the use of neomycin as a selective inhibitor of polyphosphoinositide metabolism.
Role of NADP+-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart
Authors: Sundqvist K E , Heikkilä J , Hassinen I E , Hiltunen J K
Published: August 2015

Cytosolic and mitochondrial concentrations of malate, 2-oxoglutarate, isocitrate and pyruvate in the isolated perfused rat heart were measured by non-aqueous tissue fractionation, taking the NADP-linked isocitrate dehydrogenase as indicator reactions for the free [NADPH]/[NADP+] ratios. The mass-action ratios of NADP-linked malic enzymes (EC 1.1.1.40) were found to be on the side of pyruvate carboxylation by more than one order of magnitude in both the cytosolic and the mitochondrial spaces in hearts perfused with glucose, whereas during propionate perfusion this ratio approached the equilibrium constant (Keq.) of malic enzyme. The results consequently indicate that the NADP-linked malic enzymes cannot be responsible for the feed-out (cataplerotic) reactions from the tricarboxylic acid cycle which occur during glucose perfusion. Only when other anaplerotic fluxes into the cycle are high, as during propionate oxidation, which results in accumulation of tricarboxylic acid-cycle intermediates, is a steady state reached which allows efflux via the malic enzyme.