bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of H-protein loaded with methylamine as a transient species in glycine decarboxylase reactions
Authors: Neuburger M , Jourdain A , Douce R
Published: August 2015

A three-step protocol was devised to purify H-protein, which can be readily released as a soluble protein from pea mitochondria. After the final step of purification (anion-exchange chromatography) the native enzyme was eluted as two distinct peaks at 250 and 350 mM-KCl if the lysis buffer contained glycine. Each from exhibited an identical Mr of 15000 on SDS/PAGE and they were not distinguishable by PAGE under non-denaturating conditions. Both forms catalysed the rapid fixation of [14C]bicarbonate to the carboxy group atom of glycine during the exchange reaction, whereas the reversible exchange of electrons between NADH and lipoamide bound to the H-protein in the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) was seen only with the form eluted at 350 mM-KCl. During the early steps of H-protein isolation, when P- and H-protein react together in the presence of glycine, the methylamine intermediate bound to the lipoamide of the H-protein accumulates in the medium at the expense of oxidized H-protein. Under these conditions the methylamine intermediate, which is a rather stable structure, was easily separated from the oxidized H-protein on ion-exchange chromatography. The methylamine bound to the lipoamide of the H-protein prevented the reversible exchange of electrons between NADH and lipoamide. High concentrations of glycine were required for the loading of H-protein with methylamine catalysed by a large excess of P-protein.
Regulation of liver cell volume and proteolysis by glucagon and insulin
Authors: Vom Dahl S , Hallbrucker C , Lang F , Gerok W , Häussinger D
Published: August 2015

The effects of insulin and glucagon on liver cell volume and proteolysis were studied in isolated perfused rat liver. The rate of proteolysis was assessed as [3H]leucine release from single-pass-perfused livers from rats which had been prelabelled in vivo by intraperitoneal injection of [3H]leucine. The intracellular water space was determined from the wash-out profiles of simultaneously added [3H]inulin and [14C]urea. In normo-osmotic (305 mosM) control perfusions the intracellular water space was 548 +/- 10 microliters/g wet mass (n = 44) and was increased by 16.5 +/- 2.6% (n = 6), i.e. by 85 +/- 14 microliters/g, after hypoosmotic exposure (225 mosM). Glucagon (0.1 microM) decreased the intracellular water space by 17 +/- 4% (n = 4), whereas insulin (35 nM) increased the intracellular water space by 9.3 +/- 1.4% (n = 15). Also, in isolated rat hepatocyte suspensions insulin (100 nM) caused cell swelling by 10.7 +/- 1.8% (n = 16), which was fully reversed by glucagon. In perfused liver, insulin-induced cell swelling was accompanied by a hepatic net K+ uptake (4.5 +/- 0.2 mumol/g) and an inhibition of proteolysis by 21 +/- 2% (n = 12); further addition of glucagon led to a net K+ release of 3.8 +/- 0.2 mumol/g (n = 7) and fully reversed the insulin effects on both cell volume and proteolysis. Similarly, insulin-induced cell swelling and inhibition of proteolysis were completely antagonized by hyperosmotic (385 mosM) cell shrinkage. Furthermore, cell swelling and inhibition of proteolysis after hypo-osmotic exposure or amino acid addition were reversed by glucagon-induced cell shrinkage. There was a close relationship between the extent of cell swelling and the inhibition of proteolysis, regardless of whether cell volume was modified by insulin, glucagon or aniso-osmotic exposure. The data show that glucagon and insulin are potent modulators of liver cell volume, at least in part by alterations of cellular K+ balance, and that their opposing effects on hepatic proteolysis can largely be explained by opposing effects on cell volume. It is hypothesized that hormone-induced alterations of cell volume may represent an important, not yet recognized, mechanism mediating hormonal effects on metabolism.
A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains α(2-3)-linked N-acetylneuraminic acid
Authors: Dickenson J M , Huckerby T N , Nieduszynski I A
Published: August 2015

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).
Endothelial cell growth factor and heparin regulate collagen gene expression in keloid fibroblasts
Authors: Tan E M L , Peltonen J
Published: August 2015

Keloids are benign cutaneous tumours characterized by excess deposition of collagen, specifically type I collagen. We report here that collagen biosynthesis, as measured by hydroxyproline synthesis, was markedly inhibited by 65-80% by the combination of endothelial cell growth factor (ECGF) supplement and heparin in keloid fibroblast cultures. Fibroblast cultures that were incubated with ECGF alone also demonstrated a measurable decrease of approx. 50% in collagen synthesis compared with control cultures. The inhibition of collagen synthesis was related to the down-regulation of collagen gene expression. Quantitative measurements of mRNA-cDNA hybrids revealed that the gene expression of collagen type I was decreased by more than 80% by heparin and ECGF. Markedly diminished levels of mRNA encoding collagen type I were also observed in cultures incubated with ECGF alone. The results show that ECGF and heparin elicit a negative regulatory effect on collagen production, and that this inhibition is due largely to the down-regulation of the pro-alpha 1(I) of type I collagen gene. Furthermore, ECGF has a potent suppressive effect, and heparin provides an additive effect to this inhibitory phenomenon.
Glycolipid modification of α2 interferon binding. Sequence similarity between the α2 interferon receptor and verotoxin (Shiga-like toxin) B-subunit
Authors: Lingwood C A , Yiu S K
Published: August 2015

Previous studies have implicated the glycolipid receptor for the Escherichia coli-derived verotoxin, globotriaosylceramide (Gb3; Gal alpha 1-4Gal beta 1-4Glc-ceramide), in the mechanism of alpha 2 interferon signal transduction. Comparison of the amino acid sequence of the human alpha 2 interferon receptor with that of the B (receptor-binding)-subunit of verotoxin shows three regions of similarity which may provide a structural basis for alpha 2-interferon-receptor/Gb3 interaction.
The stereochemical course of reactions catalysed by the cellobiohydrolases produced by Talaromyces emersonii
Authors: Brooks M M , Tuohy M G , Savage A V , Claeyssens M , Coughlan M P
Published: August 2015

Three forms of exocellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. CBH IA and CBH II appear to be native forms of these enzymes, while CBH IB may represent a proteolytic degradation product of the CBH IA enzyme. The hydrolysis of beta-cellobiosyl fluoride by each form was monitored by 1H-n.m.r. spectroscopy. The reactions catalysed by CBH IA and CBH IB proceed with retention of the anomeric configuration, whereas that catalysed by CBH II is one of inversion. Thus one may deduce that CBH IA (or CBH IB) and CBH II operate double and single displacement reactions respectively during catalysis of substrate. On the basis of these findings and the observed substrate specificities of the various forms, one may conclude that CBH IA (and CBH IB) is a family C enzyme, while CBH II belongs to family B [Henrissat, Claeyssens, Tomme, Lemesle & Mornon (1989) Gene 81, 83-95].
Isolation of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine from human urine
Authors: Kinuta M , Ubuka T , Yao K , Futani S , Fujiwara M , Kurozumi Y
Published: August 2015

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), a proposed precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta, Yao, Masuoka, Ohta, Teraoka & Ubuka (1991) Biochem. J. 275, 617-721], was isolated from healthy human urine by using ion-exchange column chromatography. Identification of the isolated compound with compound (I) was performed by physicochemical analyses involving i.r., m.s. and n.m.r. spectrometries as well as high-voltage paper electrophoresis, t.l.c. and paper chromatography. Compound (I) was synthesized in 80% yield by incubation of a reaction mixture containing trans-urocanic acid and 3-fold excess of cysteine at 70-75 degrees C. From these results we suggest that natural thiol compounds such as cysteine and GSH participate in the metabolism of urocanic acid, a key metabolite of L-histidine.
Rapid purification of the gastric H+/K+-ATPase complex by tomato-lectin affinity chromatography
Authors: Callaghan J M , Toh B H , Simpson R J , Baldwin G S , Gleeson P A
Published: August 2015

We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized gastric H+/K(+)-ATPase complex.
Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis
Authors: Feild J A , Reid R H , Rieman D J , Kline T P , Sathe G , Greig R G , Anzano M A
Published: August 2015

Site-directed mutants of transforming growth factor-alpha (TGF-alpha) were expressed in an Escherichia coli outer membrane protein A (ompA) expression/secretion vector under the transcriptional control of the lambda PL promoter. TGF-alpha mutant proteins were isolated from cell pellets using alkaline extraction with 0.1 M-Tris (pH 10.5). The levels of protein expression of 23 TGF-alpha mutants were comparable with those of wild-type TGF-alpha, as determined by immunoblotting and radioimmunoassay. An analysis of biological activity using as assays radioreceptor binding competition and colony formation in soft agar showed that the following mutations destroy the activity of TGF-alpha: Gly-19 to Val, Val-33 to Pro and Gly-40 to Val. Mutations of Arg-42 to Lys, Leu-48 to Ala, Tyr-38 to Trp or Phe-17 to Tyr significantly decrease, but do not destroy, biological activity when compared with the wild-type. Mutations in 14 other residues did not significantly alter receptor binding or colony-forming activity. These studies suggest that two domains localized at the surface of TGF-alpha are important in receptor binding and colony-forming activity. Domain I involves amino acid residues which include Tyr-38 and Leu-48; domain II includes residues Phe-15, Phe-17 and Arg-42.
Analysis of the five glycosylation sites of human α1-acid glycoprotein
Authors: Treuheit M J , Costello C E , Halsall H B
Published: August 2015

Orosomucoid (OMD) contains complex bi-, tri- and tetra-antennary glycan chains. Subfractionation of OMD into three molecular variants using concanavalin A lectin chromatography is based on variations in these complex structures. Standard h.p.l.c. profiles have been developed to analyse the percentage and distribution of the glycoforms present at each glycosylation site in OMD and its molecular variants. The ability to quantify the glycoforms present at each site allows us to extend the earlier results of others and resolve the remaining questions concerning the glycan structures of these variants. Most significantly, the proportions of bi-, tri- and tetra-antennary chains differ at each site for the three molecular variants. The most strongly retained variant from concanavalin A is uniquely capable of possessing biantennary chains at all five sites, whereas the unretained variant is completely devoid of biantennary chains. Only glycosylation site II of the five present is 100% biantennary in the retained and weakly retained variants. In addition, the two gene products of OMD were differentially glycosylated. Molecular masses of the glycoforms were verified by matrix-assisted u.v. laser desorption mass spectrometry. On the basis of the site distribution of oligosaccharides in the variants, efforts were made to understand the factors that control the processing of the carbohydrate chains in OMD. The results indicate that the ‘site-directed’ model of processing offers the most consistent explanation for the structures seen at the individual glycosylation sites of OMD.
Identification of the loci of the collagen-associated Ehrlich chromogen in type I collagen confirms its role as a trivalent cross-link
Authors: Kuypers R , Tyler M , Kurth L B , Jenkins I D , Horgan D J
Published: August 2015

Collagenous peptides containing the Ehrlich chromogen (EC) were selectively isolated from a tryptic digest of bovine tendon by coupling to a diazotized polyacrylamide support. The isolated p-phenol-azo-EC peptides were purified and characterized by amino acid and sequence analyses. EC occurred in stoichiometric amounts in trimeric cross-linked chains originating from the known cross-link regions of type-I collagen. The major locus of the EC was alpha 2(I)Hyl-933 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N but it was also shown to occur at the loci alpha 1(I)Hyl-87 x alpha 1(I)Lys(Hyl)-16C x alpha 1(I)Lys(Hyl)-16C and alpha 1(I)Hyl-930 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N. After sequence analyses of the C-terminal helical cross-link region alpha 2(I)928-963, corrections are presented for residues 927, 930, 932 and 933 of the bovine alpha 2(I) chain. The collagen-associated EC is postulated to be a trisubstituted pyrrole formed by the reaction of the aldehyde form of a telopeptidyl lysine residue with a bifunctional keto amino cross-link. It is also proposed that when the telopeptidyl lysine residue is hydroxylated the above reaction will result in pyridinoline formation.
Selective inhibition of protein kinase C. Effect on platelet-activating-factor-induced platelet functional responses
Authors: Murphy C T , Westwick J
Published: August 2015

The role of protein kinase C (PKC) in platelet-activating-factor (PAF)-induced platelet activation was examined by using two selective inhibitors of PKC, namely Ro 31-7549/001 and Ro 31-8220/002. Both inhibitors dose-dependently inhibited PAF-induced phosphorylation of the major 40-47 kDa protein substrate of PKC, with 50% inhibition at 4.5 microM-Ro 31-7549/001 and 0.7 microM-Ro 31-8220/002. Inhibition of PKC had no effect on maximal elevation of intracellular Ca2+ [Ca2+]i produced by either a high or a low dose of PAF, but significantly increased the duration of the Ca2+ signal and the thromboxane B2 (TxB2) generation in high-dose PAF-stimulated platelets. The inhibitors also abrogated the effect of the PKC activator phorbol 12-myristate 13-acetate on PAF-induced [Ca2+]i elevation. Sub-maximal PAF-induced dense-granule release and platelet aggregation were dose-dependently inhibited by Ro 31-7549/001 and Ro 31-8220/002. The findings suggest that endogenously activated PKC holds a bifurcating role in PAF-activated platelets, negatively affecting duration of both [Ca2+]i and TxB2 generation, and positively influencing dense-granule release and aggregation.
Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour
Authors: Eto I , Grubbs C J
Published: August 2015

Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family.
Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed ω-3 and ω-6 fatty acids
Authors: Rustan A C , Christiansen E N , Drevon C A
Published: August 2015

Rats were fed, for 3 weeks, high-fat (20% w/w) diets containing sunflower-seed oil, linseed oil or fish oil. Chow-fed rats were used as a low-fat reference. The high-fat diets markedly reduced non-fasting-rat serum triacylglycerol as compared with the low-fat reference, and the highest reduction (85%) was observed with the fish-oil group, which was significantly lower than that of the other high-fat diets. The serum concentration of phospholipids was significantly reduced (30%) only in the fish-oil-fed animals, whereas serum non-esterified fatty acids were reduced 40-50% by both the fish-oil- and linseed-oil-fed groups. The liver content of triacylglycerol showed a 1.7-fold increase with the fish-oil diet and 2-2.5-fold with the other dietary groups when compared with rats fed a low-fat diet, whereas the hepatic content of phospholipids was unchanged. Peroxisomal fatty acid oxidation (acyl-CoA oxidase) was 2-fold increased for the rats fed fish oil; however this was not significantly higher when comparison was made with rats fed the linseed-oil diet. There was no difference in phosphatidate hydrolysis (microsomal and cytosolic fractions) among animals fed the various diets. Acyl-CoA:diacylglycerol acyltransferase activity was increased by all high-fat diets, but the fish-oil-diet-fed group showed a significantly lower enzyme activity than did rats fed the other high-fat diets. A linear correlation between acyl-CoA:diacylglycerol acyltransferase activity and liver triacylglycerol was observed, and the microsomal enzyme activity was decreased 40-50% by incubation in the presence of eicosapentaenoyl-CoA. CoA derivatives of arachidonic, linolenic and linoleic acid had no inhibitory effect when compared with the control. These results indicate that dietary fish oil may have greater triacylglycerol-lowering effect than other polyunsaturated diets, owing to decreased triacylglycerol synthesis caused by inhibition of acyl-CoA:diacylglycerol acyltransferase. In addition, increased peroxisomal fatty acid oxidation and decreased availability of non-esterified fatty acids could also contribute by decreasing the amounts of fatty acids as substrates for triacylglycerol synthesis and secretion.
Glucagon, vasopressin and angiotensin all elicit a rapid, transient increase in hepatocyte protein kinase C activity
Authors: Tang E K , Houslay M D
Published: August 2015

Challenge of intact hepatocytes with one of the hormones vasopressin, angiotensin and glucagon or with the phorbol ester phorbol 12-myristate 13-acetate (PMA) led to a rapid increase in the activity of protein kinase C found in both cytosol and membrane fractions. Maximal activation by hormones occurred within 1-6 min of challenge of cells, after which activity declined. In membrane fractions protein kinase C activity return to basal levels some 15 min after exposure of cells to either angiotensin or glucagon. In cytosol fractions of cells challenged with hormones a second phase of activation ensued after about 10 min, with levels of protein kinase C activity remaining elevated above basal level 15 min afterwards. Activity changes elicited by PMA were rather different; it took about 15 min to achieve maximal activation of cytosolic protein kinase C activity. In membranes of cells challenged with PMA, an initial rapid and transient activation was followed by a sustained increase in activity occurring about 10 min after exposure of cells to this ligand. Only when hepatocytes were challenged with PMA was the translocation of protein kinase C from the cytosol to membrane fraction observed. The kinetics of PMA-induced translocation suggested that it accounted for the second phase of the increase in membrane protein kinase C activity which was unique to this ligand.
Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca2+-dependent polyphosphoinositide-specific phospholipase C
Authors: Cubitt A B , Firtel R A
Published: August 2015

We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
The γ-aminobutyric acid transporter and its interaction with taurine in the apical membrane of the bovine retinal pigment epithelium
Authors: Sivakami S , Ganapathy V , Leibach F H , Miyamoto Y
Published: August 2015

The characteristics of gamma-aminobutyric acid (GABA) uptake were investigated in apical membrane vesicles prepared from the bovine retinal pigment epithelium. An inwardly directed NaCl gradient stimulated GABA uptake markedly, and the time course of uptake exhibited an overshoot phenomenon indicating the presence of an active transport mechanism for GABA in these membranes. Other monovalent cations were not capable of substituting for Na+. In addition to this obligatory requirement for Na+, the GABA uptake also exhibited a Cl(-)-dependence, evident from the observations that the uptake was negligible in the presence of NaF or sodium gluconate in place of NaCl. NO3- and SCN- could substitute for Cl- to some extent. The uptake process was electrogenic, with a Na+/Cl-/GABA stoichiometry of 2:1:1 or 3:1:1. Substrate-specificity studies showed that the beta-amino acids such as taurine, hypotaurine and beta-alanine interacted with the GABA uptake process. Uptake of GABA could be completely inhibited by an excess of taurine and, similarly, uptake of taurine could be completely inhibited by an excess of GABA, suggesting that common transport processes operate in the uptake of these two compounds. However, a number of compounds which are specific inhibitors of GABA uptake inhibited taurine uptake only to a maximum of 50%. Kinetic analysis of GABA uptake in the concentration range 0.1-10 microM revealed that the uptake occurred via a single system and that taurine was a competitive inhibitor of this system. The Michaelis-Menten constant (Kt) for GABA was 0.94 microM and the apparent inhibition constant (Ki) for taurine was 230 microM. On the contrary, even though the kinetic analysis of taurine uptake in the concentration range 25-150 microM revealed participation of a single system in the uptake process, the inhibition of taurine uptake by GABA was not competitive. The presence of GABA decreased the maximal velocity of the taurine uptake process and also decreased the Kt for taurine. Based on these data, it is proposed that: (i) there are two distinct transport systems, namely the GABA transporter and the taurine transporter, in these membranes which accept both GABA and taurine as substrates, (ii) the affinities of these systems for taurine are very similar and cannot be kinetically distinguished under the experimental conditions employed, and (iii) the difference between the affinities of these system for GABA is much greater than for taurine.
Late steady increase in cytosolic Ca2+ preceding hypoxic injury in hepatocytes
Authors: Brecht M , Brecht C , De Groot H
Published: August 2015

Upon initiation of hypoxia, the ATP content of hepatocytes in monolayer cultures fell within 5 min from 22 to 12 nmol of ATP/10(6) cells. This decrease in ATP was not followed by early alterations in the cytosolic Ca2+ concentration; for up to 60 min it remained around 100 nM. However, after the period cytosolic free Ca2+ steadily increased, up to 400 nM. This increase began around 60 min before the cells lost their viability, and primarily resulted from an influx of extracellular Ca2+. Likewise, in experiments where the mitochondrial respiratory chain was blocked by KCN and glycolysis was blocked by iodoacetate, the ATP content fell within minutes to 10 nmol/10(6) cells, whereas the cytosolic Ca2+ concentration only began to increase 30 min later (up to 600 nM). However, also under these conditions of ‘chemical hypoxia’ this increase was clearly (about 10 min) earlier than the loss of viability.
Expression of renal organic cation transporter in Xenopus laevis oocytes
Authors: Hori R , Hirai M , Katsura T , Takano M , Yasuhara M , Kaneko S , Satoh M
Published: August 2015

The expression of the organic cation transport system of rat renal proximal tubules has been studied in Xenopus laevis oocytes injected with poly(A)+ RNA from the rat renal cortex. The effectiveness of the technique was confirmed by examining expression of the Na+/D-glucose co-transporter. Compared with water-injected and non-injected oocytes, the injection of total poly(A)+ RNA resulted in about a 3-fold increase in tetraethylammonium (TEA) uptake activity. TEA uptake by poly(A)(+)-RNA-injected oocytes was time-dependent and was inhibited by cimetidine and HgCl2, but not by p-aminohippurate. After size-fractionation on a sucrose density gradient, a 1.4-2.4 kb poly(A)+ RNA fragment was identified that expressed the organic cation transport system in oocytes. These results demonstrate that the renal organic cation transporter was expressed in oocytes and that this expression system can provide an effective assay procedure for cloning of the organic cation transporter.
An investigation of the relationships between rate and driving force in simple uncatalysed and enzyme-catalysed reactions with applications of the findings to chemiosmotic reactions
Authors: Stoner C D
Published: August 2015

Both the rate and the driving force of a reaction can be expressed in terms of the concentrations of the reactants and products. Consequently, rate and driving force can be expressed as a function of each other. This has been done for a single-reactant, single-product, uncatalysed reaction and its enzyme-catalysed equivalent using the van't Hoff reaction isotherm and Haldane's generalized Michaelis-Menten rate equation, the primary objective being explanation of the exponential and sigmoidal relationships between reaction rate and delta mu H+ commonly observed in studies on chemiosmotic reactions. Acquisition of a purely thermodynamic rate vs. driving-force relationship requires recognition of the intensive and extensive variables and maintenance of the extensive variables constant. This relationship is identical for the two reactions and is hyperbolic or sigmoidal, depending on whether the equilibrium constant is smaller or larger than unity. In the case of the catalysed reaction, acquisition of the purely thermodynamic relationship requires the assumption that the enzyme be equally effective in catalysing the forward and backward reactions. If this condition is not met, the relationship is modified by the enzyme in a manner which can be determined from the ratio of the Michaelis constants of the reactant and product. Under conditions of enzyme saturation in respect to reactant+product, the rate vs. driving-force relationship is determined exclusively by the thermodynamics of the reaction and a single kinetic parameter, the magnitude of which is determined by the relative effectiveness of the enzyme in catalysing the forward and backward reactions. In view of this finding, it is pointed out that, since the catalytic components of chemiosmotic reactions appear to be saturated with respect to the reactant-product pair that is varied in experimental rate vs. delta mu H+ determinations, and that, since many complex enzymic reactions conform to the simple Michaelis-Menten equation with respect to a single reactant-product pair when the concentrations of all other reactants and products are maintained constant, one might expect to be capable of simulating the experimental relationships simply from knowledge of the thermodynamics of the reaction and the relative effectiveness of the catalytic component in catalysing the forward and backward reactions using the simple Michaelis-Menten equation. That this expectation appears to be largely correct is demonstrated with model reactions.(ABSTRACT TRUNCATED AT 400 WORDS)
Transcriptional regulation of transferrin and albumin genes by retinoic acid in human hepatoma cell line Hep3B
Authors: Hsu S L , Lin Y F , Chou C K
Published: August 2015

Transferrin and albumin, which are both secreted from the human hepatoma cell line Hep3B, were regulated transcriptionally by retinoic acid (RA) in a dose-dependent manner. The cell growth rate was little affected under the same conditions. The treatment of Hep3B cells with RA (10 microM for 48 h) resulted in an 8-fold increase in transferrin protein synthesis, a 10-fold increase in the steady-state transferrin mRNA level, and a 5-fold increase in its transcriptional rate. The same treatment led to 4-fold decrease in albumin synthesis, as well as a 7-fold decline in the steady-state albumin mRNA level and a 4-fold decrease in the transcriptional rate. Cycloheximide and actinomycin D blocked the action of RA, suggesting that RA may regulate transferrin and albumin gene expression indirectly in human liver cells.
Human macrophages convert l-tryptophan into the neurotoxin quinolinic acid
Authors: Heyes M P , Saito K , Markey S P
Published: August 2015

Substantial increases in the concentrations of the excitotoxin and N-methyl-D-aspartate-receptor agonist quinolinic acid (QUIN) occur in human patients and non-human primates with inflammatory diseases. Such increases were postulated to be secondary to induction of indoleamine 2,3-dioxygenase in inflammatory cells, particularly macrophages, by interferon-gamma. To test this hypothesis, human peripheral-blood macrophages were incubated with L-[13C6]tryptophan in the absence or presence of interferon-gamma. [13C6]QUIN was quantified by gas chromatography and electron-capture negative-chemical-ionization mass spectrometry. [13C6]QUIN was detected in the incubation medium of both unstimulated and stimulated cultures. Exposure to interferon-gamma substantially increased the accumulation of [13C6]QUIN in a dose- and time-dependent manner. The QUIN concentrations achieved exceeded those reported in both cerebrospinal fluid and blood of patients and of non-human primates with inflammatory diseases. Macrophages stimulated with interferon-gamma may be an important source of accelerated L-tryptophan conversion into QUIN in inflammatory diseases.
Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus
Authors: Hipps D S , Perham R N
Published: August 2015

A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.
DNA methylation inhibits transcription of procollagen α2(I) promoters
Authors: Guenette D K , Ritzenthaler J D , Foley J , Jackson J D , Smith B D
Published: August 2015

Our previous studies have demonstrated that a 2-[N-(acetoxyacetyl)amino]fluorene-transformed rat epithelial-like cell line, W8, contains a transcriptionally inactive alpha 2(I) gene with a hypermethylated promoter/first-exon region. We have cloned the rat promoter/first-exon region (-211 to +207) from W8 cells and their parent cell line, K16, which expresses alpha 2(I) collagen. There were no sequence differences between the clones from the two cell lines, indicating that a mutation was not responsible for transcriptional inhibition. The alpha 2(I) rat promoters were cloned upstream of the chloramphenicol acetyltransferase gene. Both constructs were equally active in both cell lines, suggesting that trans-activating factors for alpha 2(I) transcription are present in W8 cells. Finally, methylation of plasmids at all CpG sites with SssI methylase completely inhibited transcription using alpha 2(I) promoters, but methylation did not inhibit simian-virus-40 promoter-driven transcription. Certain methylation sites partially inhibit promoter activity. An HhaI methylation site inhibited transcriptional activity of the alpha 2(I) promoter 8-fold, whereas methylation at the HpaII site in the rat alpha 2(I) promoter did not decrease transcriptional activity. This provides further evidence that methylation at specific sites in the collagen alpha 2(I) promoter is responsible for the inactivation of transcription in W8 cells.
Protein kinase C group B members PKC-δ, -ɛ, -ζ and PKC-L(η). Comparison of properties of recombinant proteins in vitro and in vivo
Authors: Liyanage M , Frith D , Livneh E , Stabel S
Published: August 2015

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.
Isolation of different high-Mr mucin species from human whole saliva
Authors: Veerman E C I , van den Keybus P A M , Valentijn-Benz M , Nieuw Amerongen A V
Published: August 2015

By using CsCl-density-gradient ultracentrifugation, two high-Mr mucin species were isolated from human whole saliva, having buoyant densities in 0.2 M-guanidinium chloride of approx. 1.56 g/ml (pool IA) and 1.48 g/ml (pool IIA). Analytical density-gradient centrifugation of submandibular, sublingual, labial and palatal saliva, followed by immunochemical analysis with anti-mucin monoclonal antibodies, indicated immunochemical and physicochemical similarities between the high-density mucins of pool IA and mucins from palatal salivary glands. Chemical analysis indicated that the putative palatal mucin was rich in sulphate, but poor in sialic acid. The lower-density mucins of pool IIA equated with the high-Mr mucins of submandibular-sublingual saliva, both immunochemically and physicochemically (buoyant density).
Identification and partial purification of the erythrocyte l-lactate transporter
Authors: Poole R C , Halestrap A P
Published: August 2015

1. Intact erythrocytes were incubated with 100 microM-4,4′-di-isothiocyanostilbene-2,2′-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig erythrocytes, were detected by this technique. In human erythrocytes, which have 10-fold less transport activity, no labelled polypeptide in this molecular mass range was detected. 2. Labelling of these 35-50 kDa polypeptides was decreased markedly in the presence of the specific inhibitors of lactate transport alpha-cyano-4-hydroxycinnamate and 4,4′-dibenzamidostilbene-2,2′-disulphonate (DBDS), which compete with DIDS for binding to the transporter. However, the weakly bound inhibitor 4,4′-dinitrostilbene-2,2′-disulphonate (DNDS) afforded little protection against labelling by DIDS. 3. The lactate transporter from rat erythrocytes was solubilized with decanoyl-N-methyl glucamide (MEGA-10) and partially purified by Mono-Q anion-exchange chromatography, with transport activity eluting at 0.1-0.15 M-NaCl. The 35-45 kDa DIDS-labelled polypeptide from rat erythrocytes was eluted in the same peak of protein as lactate transporter activity during Mono-Q chromatography. 4. These observations provide strong evidence that the lactate transporter is a polypeptide of 35-45 kDa in rat erythrocytes and of 40-50 kDa in rabbit and guinea pig erythrocytes.
Control of respiration and ATP synthesis in mammalian mitochondria and cells
Authors: Brown G C
Published: August 2015

We have seen that there is no simple answer to the question ‘what controls respiration?’ The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that oxidative phosphorylation is often close to equilibrium (apart from cytochrome oxidase) and as a consequence respiration and ATP synthesis are mainly regulated by (a) the phosphorylation potential, and (b) the NADH/NAD+ ratio. However, oxidative phosphorylation is not always close to equilibrium, at least in isolated mitochondria, and relative proximity to equilibrium does not prevent the respiratory chain, the proton leak, the ATP synthase and ANC having significant control over the fluxes. Thus in some conditions respiration rate correlates better with [ADP] than with phosphorylation potential, and may be relatively insensitive to mitochondrial NADH/NAD+ ratio.(ABSTRACT TRUNCATED AT 400 WORDS)
l-arginine transport is increased in macrophages generating nitric oxide
Authors: Bogle R G , Baydoun A R , Pearson J D , Moncada S , Mann G E
Published: August 2015

Transport of L-arginine and nitrite production were examined in the murine macrophage cell line J774. Bacterial lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production, which was further increased in the presence of interferon-gamma. Nitrite synthesis was absolutely dependent on extracellular L-arginine and inhibited in the presence of L-lysine or L-ornithine. In unactivated J774 cells L-arginine transport was saturable, with an apparent Km of 0.14 +/- 0.04 mM and Vmax. of 15 +/- 2 nmol/h per 10(6) cells. LPS (1 microgram/ml) induced a time-dependent stimulation of L-arginine transport, and after 24 h the Vmax. increased to 34 +/- 2 nmol/h per 10(6) cells. These findings indicate that activation of J774 cells with LPS produces an increase in both L-arginine transport and nitrite synthesis. The elevated rate of L-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization of L-arginine for the generation of NO.
Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes
Authors: Graziani A , Ling L E , Endemann G , Carpenter C L , Cantley L C
Published: August 2015

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.