bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics
Authors: Vasilyeva E A , Minkov I B , Fitin A F , Vinogradov A D
Published: August 2015

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.
Studies on the efflux of metalloporphyrin from isolated rat liver mitochondria. Effect of respiratory substrates and metabolic inhibitors
Authors: Husby P , Romslo I
Published: August 2015

Intramitochondrially synthesized Co-deuteroporphyrin is released to the incubation medium at a rate inversely correlated to the energy state of the mitochondria; i.e. the rate of efflux increases when substrate is depleted, respiration inhibited or the mitochondria are uncoupled. The efflux of Co-deuteroporphyrin from mitochondria remains low as long as the residual membrane potential is above one-third that of maximally energized mitochondria. Globin enhances the efflux of Co-deuteroporphyrin not only from mitochondria depleted of substrates [Husby & Romslo (1980) Biochem. J. 188, 459-465], but also from maximally energized mitochondria. The results provide further evidence for a co-operative mechanism between the mitochondria and their surroundings for the mobilization of metalloporphyrin from mitochondria.
Muscle protein synthesis in the streptozotocin-diabetic rat. A possible role for corticosterone in the insensitivity to insulin infusion in vivo
Authors: Odedra B R , Dalal S S , Millward D J
Published: August 2015

The effect of insulin infusion in vivo on muscle protein synthesis was investigated in rats. In 10-days-streptozotocin-diabetic rats infused in vivo with amino acids and glucose, the rate of protein synthesis per unit of RNA (RNA activity) was markedly decreased. Pre-treatment with large doses of insulin at 17 and 1 h before the infusion fully restored RNA activity to normal. Infusion of insulin for 6 h with amino acids and glucose did not restore RNA activity to normal in the diabetic rats. However, in diabetic-adrenalectomized rats similar infusions of insulin fully restored RNA activity to normal. Measurements of plasma corticosterone concentrations indicated a 50% increase in the diabetic rats. Since pre-treatment with corticosterone suppressed the stimulatory effect of insulin infusion on RNA activity in adrenalectomized rats, and since corticosterone treatment for 6 days suppressed RNA activity even though insulin concentrations were elevated, it is suggested that increased concentrations of corticosterone are responsible for the lag in response to insulin in the diabetic rat. This means that the catabolic effects of glucocorticoids must be also considered together with the catabolic effect of insulin lack in diabetes.
Reduction of membrane-bound dopamine β-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane
Authors: Grouselle M , Phillips J H
Published: August 2015

Resealed bovine chromaffin-granule ‘ghosts’ were used for assaying the membrane-bound form of dopamine beta-hydroxylase. Hydroxylation of the substrate tyramine is dependent on its accumulation within the ‘ghosts’, where the active site of the enzyme is located. Free tyramine in the medium is at a low concentration, low ionic strength and a relatively high pH (7.0), so that even in the presence of a reducing agent (co-reductant) the unaccumulated amine is hydroxylated at a negligible rate. ‘Ghosts’ contain an endogenous co-reductant, which is shown to be catecholamine remaining in the membrane itself after granule lysis. Catecholamine that is free in solution in the medium or in the interior of the ‘ghosts’ is not effective as co-reductant, nor is ascorbate, in contrast with the situation with soluble dopamine beta-hydroxylase. Ferrocyanide is an active co-reductant, however, giving a hydroxylation rate approximately equal to the tyramine accumulation rate: it does not enter the ‘ghosts’, nor does the enzyme appear to utilize ferrocyanide sealed inside the ‘ghosts’. A mechanism must therefore exist for transferring electrons across the membrane from the cytoplasmic surface to the matrix surface. NADH is not an electron donor for the enzyme, nor is cytochrome b-561 involved.
Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with l-histidine
Authors: Glennon Jeremy D. , Sarkar Bibudhendra
Published: August 2015

Detailed studies are reported on the Ni(II)-binding site of human serum albumin (HSA) and the results are compared with those obtained from the N-terminal native-sequence peptide, l-aspartyl-l-alanyl-l-histidine N-methylamide (Asp-Ala-His-NHMe). Equilibrium dialysis of HSA and Ni(II) in 0.1m-N-ethylmorpholine/HCl buffer, pH 7.53, demonstrates a specific Ni(II)-binding site on the protein. l-Histidine, the low-molecular-weight Ni(II)-binding constituent of human serum, is shown to have a greater affinity for Ni(II) than does HSA. A small but significant amount of ternary complex HSA–Ni(II)–l-histidine is also present in the equilibrium mixture containing the three components. The log (association constant) values for the binary and ternary Ni(II) complexes are 9.57 and 16.23 respectively. The complex equilibria between Asp-Ala-His-NHMe and Ni(II) have been investigated by analytical potentiometry in aqueous solution (0.15m-NaCl, 25°C). Several species, including MA, MA2, MH−2A, and MH−1A2 [where M and A represent Ni(II) ion and anionic peptide respectively], were detected in the system, MH−2A being the major complex species. Equilibrium studies involving Asp-Ala-His-NHMe, Ni(II) and l-histidine reveal the presence of a ternary complex MH−1AB (where B represents anionic l-histidine) at physiological pH. Detailed studies of visible-absorption spectra of HSA in the presence of Cu(II) and Ni(II) reveal that the two metal ions bind HSA at the same site. The visible-absorption spectrum of Ni(II)–HSA complex shows a highly absorbing peak at 420nm (εmax. = 137; with shoulder at 450–480nm) characteristic of a square planar or square pyramidal co-ordination arrangement about the metal ion. Similar visible-absorption characteristics were observed for the major species MH−2A in the Asp-Ala-His-NHMe–Ni(II) system (λmax. = 420nm; εmax. = 135; with shoulder at 450–480nm). The combination of experimental results from the protein studies and the peptide analyses provides strong evidence for the structure of the Ni(II)-binding site of HSA as one that involves the α-amino nitrogen atom, two deprotonated peptide nitrogen atoms, the imidazole nitrogen atom and the side-chain carboxy group of the aspartic acid residue. On the basis of the results obtained from the individual ternary systems involving protein and peptide, a mechanism for the transportation of Ni(II) in the serum is proposed.
Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed)
Authors: Barbieri L , Aron G M , Irvin J D , Stirpe F
Published: August 2015

1. The pokeweed antiviral protein, previously identified in two forms (PAP and PAP II) in the leaves of Phytolacca americana (pokeweed) [Obrig. Irvin & Hardesty (1973) Arch. Biochem. Biophys. 155, 278-289; Irvin, Kelly & Robertus (1980) Arch. Biochem. Biophys. 200, 418-425] is a protein that prevents replication of several viruses and inactivates ribosomes, thus inhibiting protein synthesis. 2. PAP is present in several forms in the seeds of pokeweed. One of them, which we propose to call ‘pokeweed antiviral protein from seeds’ (PAP-S) was purified in high yield (180 mg per 100 g of seeds) by chromatography on CM-cellulose, has mol.wt. 30 000, and is similar to, but not identical with. PAP and PAP II. 3. PAP-S inhibits protein synthesis in a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of 1.1 ng/ml (3.6 × 10(-11) M), but has much less effect on protein synthesis by whole cells, with an ID50 of 1 mg/ml (3.3 × 10(-5) M), and inhibits replication of herpes simplex virus type 1.
The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3
Authors: Levison P R , Tomalin G
Published: August 2015

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.
Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli
Authors: Hawkins A R , Reinert W R , Giles N H
Published: August 2015

1. Neurospora crassa catabolic dehydroquinase has been purified from N. crassa and Escherichia coli. 2. Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers. 3. Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N. crassa is expressed with fidelity in E. coli.
A complete separation of dimethylaminoazobenzenesulphonyl-amino acids. Amino acid analysis with low nanogram amounts of polypeptide with dimethylaminoazobenzenesulphonyl chloride
Authors: Chang J Y , Knecht R , Braun D G
Published: August 2015

A chromatographical system has been developed to give a complete baseline separation of all dimethylaminoazobenzenesulphonyl (DABS)-amino acids for high-sensitivity amino acid analysis [Chang, Knecht & Braun (1981) Biochem. J. 199, 547-556]. The system, which uses a Merck RP-18 column with phosphate buffer (12 mM, pH 6.5)/acetonitrile mixture, allows reliable analysis of DABS-amino acids at the 1-2 pmol level. The accuracy of this new system is demonstrated by the composition analysis of two immunoglobulin light chains (214 amino acid residues) with differences at only three amino acid residue positions.
The turnover of L-type pyruvate kinase in cultured rat hepatocytes
Authors: Poole Graeme P. , Bloxham David P.
Published: August 2015

Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-3H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [3H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t½ (half-time) 4.9h and a duration of 8–10h was followed by a phase with t½ 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1μm-insulin to the culture medium increased the t½ of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.
A serum protein inhibitor of acid lipase and its possible role in lipid accumulation in cultured fibroblasts
Authors: Gorin E , Gonen H , Dickbuch S
Published: August 2015

Histochemical examination of L929 fibroblasts indicates massive accumulation of intracellular lipids in cells grown in medium supplemented with 10% calf serum. The present study suggests that the accumulation of triacylglycerols in these cells may be due to the inhibition of acid lipase activity by a serum component present in the culture medium. This is based on the following observations. (a) Acid lipase appears to be the major intracellular enzyme responsible for triacylglycerol catabolism in L929 cells. (b) The acid lipase is strongly inhibited by either human of calf serum. Several lines of evidence show that the inhibitor is a serum protein: it is heat-labile, non-dialysable and is destroyed by trypsin. It is present mainly in Cohn's fraction IV and has mol.wt. approx. 50000. (c) Lipid accumulation in intact cells is reduced when cells are grown on a limited supply of serum (2%) and is elevated by the addition of Cohn's fraction IV, freed of lipoproteins, to the growth medium.
Regulation of acetyl-CoA carboxylase in rat mammary gland. Effects of starvation and of insulin and prolactin deficiency on the fraction of the enzyme in the active form in vivo
Authors: McNeillie Elizabeth M. , Zammit Victor A.
Published: August 2015

The ‘initial’ (I), endogenous phosphatase-activated (A) and citrate-activated (C) activities of acetyl-CoA carboxylase were measured in mammary-gland extracts of pregnant and lactating rats. There was a 10-fold increase in the A and C enzyme activities in the transition from early to peak lactation [cf. data of Mackall & Lane (1977) Biochem. J.162, 635–642], but there was no significant increase in the ratio of the initial activity to the A and C activities of the enzyme. Starvation (24h) or short-term (3h) streptozotocin-induced diabetes both resulted in a 40% decrease in I/A and I/C activity ratios. In starvation this was accompanied by a decrease in the absolute values of the A and C activities such that the initial activity in mammary glands of starved animals was 45% that in glands from fed animals. Insulin treatment of starved or diabetic animals 60min before killing increased the I activity without affecting the A or C enzyme activities. Removal of the pups for 24h from animals in peak lactation (weaning) resulted in a marked but similar decrease in all three activities such that, although the initial activity was only 10% of that in suckled animals, the I/A and I/C activity ratios remained high and unaltered. Inhibition of prolactin secretion by injection of 2-bromo-α-ergocryptine gave qualitatively similar results to those during weaning. Simultaneous administration of ovine prolactin completely prevented the effects of bromoergocryptine. It is suggested that the initial activity of acetyl-CoA carboxylase in rat mammary gland is regulated by at least two parallel mechanisms: (i) an acute regulation of the proportion of the enzyme in the active state and (ii) a longer-term modulation of enzyme concentration in the gland. Insulin appeared to mediate its acute effects through mechanism (i), whereas prolactin had longer-term effects on enzyme concentration in the gland. A comparison of initial enzyme activities (I) obtained in the present study with rates of lipogenesis measured in vivo [Agius & Williamson (1980) Biochem. J.192, 361–364; Munday & Williamson (1981) Biochem. J.196, 831–837] gave good agreement between the two sets of data for all conditions studied except for 24h-starved and streptozotocin-diabetic animals. It is suggested that acetyl-CoA carboxylase activity is rate-limiting for lipogenesis in the mammary gland in normal, fed, suckled or weaned animals but that in starved and short-term diabetic animals changes in the activity of the enzyme by covalent modification alone may not be sufficient to maintain the enzyme in its rate-limiting role.
Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein
Authors: Fry S C
Published: August 2015

1. Cell-wall hydrolysates from calli of all higher plants tested contained a new phenolic amino acid for which the trivial name isodityrosine is proposed. Isodityrosine was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a diphenyl ether bridge. 2. The amount of isodityrosine in sodium dodecyl sulphate-insoluble cell-wall preparations was proportional to the amount of hydroxyproline. 3. Acidified chlorite split the diphenyl ether bridge of isodityrosine, and concomitantly solubilized the cell-wall glycoprotein. 4. Dithiothreitol inhibited isodityrosine synthesis in vivo, and suppressed in parallel the covalent binding of newly synthesized protein in the cell wall. 5. It is suggested that isodityrosine is an inter-polypeptide cross-link responsible for the insolubility of plant cell-wall glycoprotein.
Identification of metallothionein in parenchymal and non-parenchymal liver cells of the adult rat
Authors: Sciortino C V , Failla M L , Bullis D B
Published: August 2015

Parenchymal and non-parenchymal cells were isolated from the livers of control, starved, Zn2+-injected and Cd2+-injected rats. Parenchymal cells were prepared by differential centrifugation after perfusion of the liver with collagenase. Non-parenchymal cells were separated from parenchymal cells by unit-gravity sedimentation and differential centrifugation. Yields of 2 × 10(8) non-parenchymal cells with greater than 95% viability and less than 0.2% contamination with parenchymal cells were obtained without exposing cells to Pronase. Metallothioneins-I and -II were identified in parenchymal cells and non-parenchymal cells from Zn2+-treated rats. The metallothionein contents of parenchymal cells, non-parenchymal cells and intact liver were quantified by a competitive 203Hg-binding assay. Administration of heavy-metal salts significantly increased the metallothionein content of both cell populations, although the concentration of the protein was approx. 2.5-fold greater in parenchymal cells than in non-parenchymal cells. Overnight starvation increased the metallothionein content of parenchymal cells without altering that of non-parenchymal cells. The potential significance of this differential response by different liver cell types with regard to the influence of Zn2+ on stress-mediated alterations in hepatic metabolism is discussed.
Superoxide dismutase, glutathione peroxidase and catalase in developing rat brain
Authors: Mavelli I , Rigo A , Federico R , Ciriolo M R , Rotilio G
Published: August 2015

The specific activities of Cu, Zn- and Mn-superoxide dismutases, of glutathione peroxidase and of catalase, the enzymes considered to be specifically involved in the defence of the cell against the partially reduced forms of oxygen, were determined as the function of postnatal age in the early (up to 60 days) period of rat brain development. The enzymes were assayed in the cytoplasmic fraction, in the crude mitochondrial fraction including peroxisomes, and in the mitochondria. The results show that the temporal changes of these enzymes cannot be correlated with each other, thus indicating that they do not concertedly parallel the increasing activity of aerobic brain metabolism during development. Specifically the cytoplasmic fraction shows a gradual increase of the Cu, Zn-superoxide dismutase activity with age, whereas the glutathione peroxidase activity is constant from birth. Furthermore the increase of the mitochondrial Mn-superoxide dismutase as a function of postnatal age is more remarkable than that of the cytoplasmic Cu, Zn-enzyme. Higher activities of catalase in adult animals are detectable only in the subcellular fraction containing peroxisomes, because of the modest catalase activity of the brain. These results indicate independent regulation of the expression of these enzyme activities in the process of brain differentiation and point to a relative deficiency of enzymic protection of the brain differentiation and point to a relative deficiency of enzymic protection of the brain against potentially toxic oxygen derivatives. This situation is similar to the pattern already described in the rat heart and in rat and mouse ascites-tumour cells, at variance with the much more efficient enzyme pattern present in rat hepatocytes.
Immunological and kinetic properties of pyruvate kinase in rat pancreatic islets
Authors: Chatterton Tracey A. , Reynolds C. Hugh , Lazarus Norman R. , Pogson Christopher I.
Published: August 2015

Pyruvate kinase in rat pancreatic islets was characterized immunologically and kinetically. It is concluded that this activity is predominantly if not totally of the M2 type.
Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen
Authors: Lebeau M C , Massol N , Baulieu E E
Published: August 2015

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13-14 S and 7-8 S. After repeated injections of oestradiol benzoate, the 13-14 S peak increased more than the 7-8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13-14 S and 7-8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13-14 S oestrogen binding sites than did progesterone alone. The presence of a 13-14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.
Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme
Authors: Gold E W
Published: August 2015

Human liver hyaluronidase was purified to homogeneity by (NH4)2SO4 fractionation, chromatography on hydroxyapatite and DEAE-cellulose, and preparative disc polyacrylamide-gel electrophoresis. The enzyme had a pH optimum of 3.8-4.0, a molecular weight (determined by gel filtration) of 76000, and a Km of 0.05 mg/ml for purified human umbilical-cord hyaluronic acid. It generally resembled hyaluronidases studied in other tissues which are believed to be lysosomal, but shared a number of characteristics with a partially purified bovine testicular hyaluronidase. Neither enzyme exhibited inhibition by high concentrations of substrate, but both were competitively inhibited by dermatan sulphate and keratan sulphate. Both enzymes exhibited increased activity in the presence of albumin, probably owing to an increased susceptibility of substrate to enzyme action. The liver enzyme was inhibited by NaCl, but the testicular enzyme exhibited an increase in activity in the presence of the salt which was similar to the effect observed with albumin. The different response toward Cl- ion appeared to be the most significant difference between the two enzymes.
Purification and structural studies on the complement-system control protein β1H (Factor H)
Authors: Sim R B , DiScipio R G
Published: August 2015

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.
Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa
Authors: Savany A , Cronenberger L
Published: August 2015

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.
A general method for affinity purification of complement component C3b using factor H-sepharose
Authors: Scott J D , Fothergill J E
Published: August 2015

Complement component C3b has been purified from human, rabbit and bovine serum by affinity chromatography on human factor H-Sepharose after preliminary fractionation by poly(ethylene glycol) and DEAE-Sepharose. The yields are high (35-40%) and the whole process is rapid (3 days). Binding of C3b to factor H-Sepharose is equimolar, has a sharp optimum pH at 7.6 and is quite sensitive to ionic strength.
The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations
Authors: Davies D D , Kenworthy P
Published: August 2015

The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the ‘B’ methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits ‘A’ stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the ‘B’ to the ‘A’ side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is ‘B’-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the ‘B’ side. A further increase in the removal of hydrogen from the ‘B’ side of NADPH could be the kinetic isotope effect that is observed when [‘A’-3H]NADPH is the reductant, the H-C bond being cleaved 2.3 times faster than the 3H-C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a ‘B’-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.
The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin
Authors: Heaphy S , Williams J
Published: August 2015

Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain.
Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat
Authors: Stipanuk M H , Beck P W
Published: August 2015

The contribution of cystathionine gamma-lyase, cystathionine beta-synthase and cysteine aminotransferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydration in rat liver and kidney was assessed with four different assay systems. Cystathionine gamma-lyase and cystathionine beta-synthase were active when homogenates were incubated with 280 mM-L-cysteine and 3 mM-pyridoxal 5′-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160 mM-L-cysteine, 2 mM-pyridoxal 5′-phosphate, 3 mM-2-oxoglutarate and 3 mM-dithiothreitol. At more-physiological concentrations of cysteine (2 mM) cystathionine gamma-lyase and cystathionine beta-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine gamma-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO4(2-) or [35S]taurine formed from labelled dietary cysteine.
The effects of glucose, acetate, lactate and insulin on protein degradation in the perfused rat heart
Authors: Sugden P H , Smith D M
Published: August 2015

Rat hearts were perfused as working preparations by the method of Taegtmeyer, Hems & Krebs [(1980 Biochem. J. 186, 701-711]. In the presence of glucose, insulin significantly inhibited protein degradation at concentrations as low as 50 mu units/ml. Acetate or lactate, when present either as sole fuel for contraction or in combination with glucose, did not inhibit protein degradation. Insulin inhibition or protein degradation was decreased with either lactate as sole fuel. We suggest that the inhibition of protein degradation occurs over the normal range of plasma concentrations of insulin present in vivo and that the presence of glucose may be at least in part necessary for this effect of insulin.
Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells
Authors: Majamaa K , Myllylä R , Alitalo K , Vaheri A
Published: August 2015

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.
Two new low-molecular-weight acidic proteins from calf thymus nuclei that resemble HMG (high-mobility-group) proteins 14 and 17
Authors: Cary P D
Published: August 2015

Two new, closely similar, acidic proteins were extracted and purified from calf thymus and designated AP-X and AP-Y (acidic proteins X and Y). They contain about 33% acidic residues, mostly non-amidated, and 20% lysine, but no arginine, tyrosine, histidine or tryptophan. There is a single phenylalanine residue in a molecular weight of approx. 5000. Circular dichroism and proton nuclear magnetic resonance show that they do not take up secondary or tertiary structure in free solution, as expected from the low content of hydrophobic amino acids. They appear structurally related to the high-mobility-group proteins HMG 14 and 17. Controlled extraction experiments indicate that proteins AP-X and AP-Y are at least partially located in the calf thymus nucleus.
The post-translational proteolysis of the subunits of vicilin from pea (Pisum sativum L.)
Authors: Gatehouse J A , Lycett G W , Croy R R D , Boulter D
Published: August 2015

Tryptic-peptide profiles and amino acid sequencing of purified pea (Pisum sativum L.) vicilin subunits were used to show that their sequences were interrelated. Comparison with the nucleotide sequence of a cloned vicilin complementary DNA (mRNA) showed that all vicilin subunits could be derived from 50 000-Mr precursors containing up to two sites for post-translational proteolytic cleavage, and allowed these subunits to be located relative to the precursor.
Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells
Authors: Ramachandran C K , Gray S L , Melnykovych G
Published: August 2015

The effect of the addition of 1 microM-dexamethasone on the temporal sequence of the glycosylation process has been studied in HeLa S3G cells. In the presence of delipidized serum, dexamethasone caused an increase in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and an accelerated synthesis of dolichols. These events were followed by an increase in the transfer of mannose from GDP-mannose to mannolipid. An increase in the rate of synthesis of lipid-linked oligosaccharides and a stimulation of glycosylation were observed in cells grown in the presence of delipidized serum in the culture medium. The data are consistent with the view that cellular syntheses of lipids and glycoproteins are co-ordinately controlled.
Underestimation of metabolic rates owing to reincorporation of 14CO2 in the perfused rat liver
Authors: Tomera John F. , Goetz Patrick G. , Rand William M. , Brunengraber Henri
Published: August 2015

14CO2 production by perfused rat livers was simulated by infusing NaH14CO3 into the perfusate. Recovery of label as 14CO2 gas + perfusate bicarbonate was 45–85%. Rates of 14CO2 exchange in the liver are 3–70 times greater than net rates of CO2 production. Therefore 14CO2 reincorporation can lead to significant underestimations of rates of oxidation of 14C-labelled substrates in liver.
Intracellular inactivation, reactivation and dynamic status of prostate androgen receptors
Authors: Rossini Gian Paolo , Liao Shutsung
Published: August 2015

The dynamic status of the androgen receptor in prostate cells was studied by incubation of rat ventral prostate with radioactive 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) in the presence and absence of respiratory poisons and inhibitors of protein and RNA synthesis and also by isotope chasing of the radioactive androgen–receptor complexes. The androgen receptor in the prostate appears to go through a dynamic process of recycling between the cytoplasm and the nucleus as well as an inactivation process. The radioactive androgen–receptor complex, however, is maintained at a constant level for at least 2h during incubation at 37°C, even in the absence of new protein synthesis, suggesting that early androgen actions may not require a depletion of a major portion of cellular receptor. In the presence of 2,4-dinitrophenol, the androgen receptor is rapidly deactivated (half life, 2min). The inactive receptor can be reactivated efficiently by an energy-dependent process, even in the absence of protein synthesis. Receptor binding of androgen and nuclear chromatin binding of the androgen–receptor complex are fast processes; half-maximum binding can be achieved within 1 and 10min respectively. On the contrary, the overall process of the release of the receptor complex from nuclear chromatin and its reappearance in the cytosol fraction has a long half life of about 70min. This slow process may reflect the involvement of the steroid–receptor complex in a time-consuming mechanism that is essential for hormone responses. Actinomycin D can increase the nuclear receptor level by 50% or more. This increase is not due to a decrease in the rate of receptor release from nuclei or to inhibition of DNA degradation by the antibiotic.
The degradative pathway of the s-triazine melamine. The steps to ring cleavage
Authors: Jutzi K , Cook A M , Hütter R
Published: August 2015

1. The degradative pathway of melamine (1,3,5-triazine-2,4,6-triamine) was examined in Pseudomonas sp. strain A. 2. The bacterium grew with melamine, ammeline, ammelide, cyanuric acid or NH+4 as sole source of nitrogen, and each substrate was entirely metabolized. Utilization of ammeline, ammelide, cyanuric acid or NH+4 was concomitant with growth. But with melamine as substrate, a transient intermediate was detected, which was identified as ammeline by three methods. 3. Enzymes from strain A were separated by chromatography on DEAE-cellulose, and four activities were examined. 4. Melamine was converted stoichiometrically into equimolar amounts of ammeline and NH+4. 5. Ammeline was converted stoichiometrically into equimolar amounts of ammelide and NH+4; ammelide was identified by four methods. 6. Ammelide was converted stoichiometrically into equimolar amounts of cyanuric acid and NH+4; cyanuric acid was identified by four methods. 7. Cyanuric acid was converted by an enzyme preparation into an unidentified product with negligible release of NH+4. 8. The specific activities of the degradative enzymes (greater than or equal to 0.3 mkat/kg of protein) were high enough to explain the growth rate of the organism. 9. The bacterium converted 0.4 mM-melamine anaerobically into 2.3 mM-NH+4. 10. Two other pseudomonads and two strains of Klebsiella pneumoniae were also examined, with similar results. 11. The degradative pathway of melamine appears to be hydrolytic, and proceeds by three successive deaminations to cyanuric acid, which is further metabolized.
Nuclear interactions of retinoic acid-binding protein in chemically induced mammary adenocarcinoma
Authors: Mehta R G , Cerny W L , Moon R C
Published: August 2015

Cellular retinoic acid-binding protein (CRABP) was detected in the nuclear fraction of N-methyl-N-nitrosourea-induced mammary cancers after the incubation of cytosol containing [3H]retinoic acid (RA)-bound CRABP with isolated nuclei. CRABP extracted from the nuclei in buffer containing 0.4 M-KCl sedimented as a 2 S component when subjected to sucrose-density-gradient analysis. [3H]RA-CRABP was found to be a prerequisite for the detection of nuclear binding, since the incubation of isolated nuclei or 0.4 M-KCl extract of the nuclei with [3H]RA did not result in any significant binding. Incubation of [3H]RA-CRABP at 25 or 30 degrees C before incubation with the nuclei neither altered the sedimentation coefficient nor enhanced the nuclear binding compared with 0 degrees C incubation. The tumour nuclei contained a saturable number of binding sites with a dissociation constant of 1.6×10(-9) M. These results indicate that the action of retinoic acid in the target organ may be mediated by its interaction with the nuclei.
Characterization of isoprotein patterns in tissue extracts and isolated samples of metallothioneins by reverse-phase high-pressure liquid chromatography
Authors: Klauser S , Kägi J H R , Wilson K J
Published: August 2015

Reverse-phase high-pressure (‘performance’) liquid chromatography was used to characterize the isometallothioneins in preparations isolated from tissues of a variety of animals by ‘conventional’ chromatographic methods. The resolution was such that isoproteins differing by a single serine leads to leucine difference in 61 residues could be easily separated. Yields from the reverse-phase support were typically 60-70% for the isoproteins. Comparisons of isometallothionein patterns after Cd2+-induction in rabbits indicated that total metallothionein concentrations were about 4-fold higher in liver than kidney extracts from the same animal. In the extracts a minimum of four and six isometallothionein peaks were detected in kidney and liver respectively. Under acidic conditions, where the metals are removed from the protein, the chromatographic properties, i.e. hydrophobicities, of the isoproteins from kidney were identical with those of four of those found in liver. Although the same peaks appeared in tissue extracts from individual animals, concentration differences were apparent. Remarkably, no differences were observed between the isoprotein patterns of liver or kidney as a consequence of either Cd2+- or Zn2+-induction. Chromatography of the metal-containing forms at neutral pH in Tris buffer indicated that the relative ratios of the complexed metal ions in the isoproteins were found to be effectively identical, not only before and after chromatography, but also within the separated forms from a single tissue source.
The alpha particulate liver glycogen. A morphometric approach to the kinetics of its synthesis and degradation
Authors: Devos P , Baudhuin P , Van Hoof F , Hers H G
Published: August 2015

Electron micrographs of rat hepatocytes with a glycogen content between 0.36 and 2.55% (w/w) were submitted to morphometrical analysis. From the number and size of glycogen profiles, the distribution of radius and volume of glycogen alpha particles were computed. The 7-fold difference in glycogen content was accompanied by an only 1.8-fold increase in the mean volume of the particles while their number increased by a factor of 4. On the basis of these observations, it is proposed that the population of glycogen particles can be divided in two groups. The first one is made of growing particles, still associated with glycogen synthase; they are the only particles present at low glycogen concentration and their number is limited. Application of a simple mathematical model allows to estimate their number in hepatocytes as 49×10(12) particles . ml-1. The second group is made of glycogen particles which have reached their maximal size and the number of which is in principle unlimited. The maximal particle size is estimated to be 0.36×10(-15) ml, corresponding to an average molecular weight of 178×10(6). The average molecular weight of glycogen, as measured from the actual size of the particles, varied from 89×10(6) to 161×10(6).
The mechanism of thermal degradation of a high-molecular-weight glycoprotein complex from bovine cervical mucus
Authors: Meyer F A , Paradossi G
Published: August 2015

Gel-like oestrus bovine cervical mucus can be brought to the point of dissolution by thermal treatment. The glycoprotein complex so produced was isolated on CsCl density gradients, and found to be of a size comparable with that of a complex purified from mucus that had been brought to the point of dissolution by mild mechanical stirring. The latter material (GP-S) had a mol.wt. of 15.9×10(6) and was used to study further the effect of thermal treatment. Time and temperature lead to a gradual breakdown of GP-S, which is characterized by a single activation energy of 93.3 kJ/mol (22.3 kcal/mol) over the temperature range of 21-99 degrees C. The process responsible is thermal hydrolysis of peptide bonds, particularly next to aspartic acid residues. This conclusion is consistent with the appearance of aspartic acid as a new N-terminal amino acid and the activation energy of the process. After thermal degradation there is an increase in the buoyant density of GP-S and a change in the amino acid composition. These findings were found to be consistent with the loss of the naked peptide region and the preponderance of aspartic acid residues in this region. Thermal degradation therefore does not involve dispersion of non-covalent bonds, and indeed GP-S is quite unaffected by media commonly used to disperse such bonds.
Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine-Sepharose affinity chromatography
Authors: Head J F , Spielberg S , Kaminer B
Published: August 2015

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.