bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin
Authors: Minocherhomjee Arda-e-Viraf , Roufogalis Basil D.
Published: August 2015

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca2++Mg2+-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2μg/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca2++Mg2+-stimulated ATPase by increasing the Ca2+-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca2++Mg2+-dependent ATPase in addition to its effect on the Ca2+-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca2++Mg2+-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca2+ concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5μm-cyclic AMP inhibited Ca2++Mg2+-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca2++Mg2+-stimulated ATPase. Secondly, trifluoperazine (20μm) antagonized the stimulation of Ca2++Mg2+-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca2++Mg2+-stimulated ATPase by PKI.
Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline
Authors: Laurent G J
Published: August 2015

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.
Superoxide-dependent formation of hydroxyl radicals and lipid peroxidation in the presence of iron salts. Detection of ‘catalytic’ iron and anti-oxidant activity in extracellular fluids
Authors: Gutteridge John M. C. , Rowley David A. , Halliwell Barry
Published: August 2015

Synovial fluid from rheumatoid patients and normal cerebrospinal fluid contains micromolar concentrations of non-protein-bound iron salts that can promote lipid peroxidation and also the superoxide-dependent formation of hydroxyl radicals from hydrogen peroxide. These iron catalysts of oxygen radical reactions cannot be detected by conventional assays unless interfering high-molecular-weight substances, probably proteins, are removed by ultrafiltration or inactivated by exposure to low pH values. The bleomycin assay for ‘catalytic’ iron [Gutteridge, Rowley & Halliwell (1981) Biochem. J.199, 263–265] does not suffer from these artifacts.
The effect of β-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ
Authors: Davis R J , Martin B R
Published: August 2015

1. The accumulation of [3H]methyltriphenylphosphonium by isolated fat-cells was used to estimate the membrane potential of mitochondria in situ. 2. Adrenaline caused a large decrease in the accumulation of [3H]methyltriphenylphosphonium. Mitochondria in fat-cells incubated in the presence of adrenaline had a very low calculated membrane potential. This effect was also given by isoprenaline (a beta-adrenergic agonist) and was blocked by propranolol (a beta-adrenergic antagonist). 3. The effect of isoprenaline could be partially antagonized by the use of media with high albumin concentrations. Addition of sodium oleate to saturate the fatty acid-binding sites on the albumin reversed this antagonism. 4. It is proposed that the decrease in the calculated mitochondrial membrane potential is due to the uncoupling effect of the non-esterified fatty acids released by the stimulation of lipolysis observed in the presence of beta-adrenergic agonists.
Characterization of the molybdate-stabilized glucocorticoid receptor from rat thymus
Authors: Weatherill P J , Bell P A
Published: August 2015

The untransformed glucocorticoid receptor of rat thymus cytosol was characterized in the form of its complex with [1,2,4-3H]triamcinolone acetonide by ion-exchange chromatography and by gel filtration and sucrose-density-gradient ultracentrifugation at different ionic strengths. Molybdate (10 mM) was present throughout all experimental procedures and prevented receptor inactivation and degradation as well as transformation. At low ionic strength the molybdate-stabilized steroid-receptor complex was detected as a single highly asymmetric entity with a Stokes radius of 5.85 nm, a sedimentation coefficient of 9.6 S and an apparent molecular weight of 236 000. This form was converted into a smaller, even more asymmetric, form in increasing proportion as the ionic strength was increased. In the presence of 0.4 M-KCl, the smaller form had a Stokes radius of 4.95 nm, a sedimentation coefficient of 4.6 S and an apparent molecular weight of 95 500. It is concluded that the glucocorticoid-receptor complex exists at low ionic strengths as a homodimer or as a heterodimer in which only one subunit possesses a steroid-binding site, and that the process of dissociation into subunits brought about by increasing the ionic strength is a process distinct from, but possibly preceding, the transformation phenomenon responsible for conferring DNA-binding properties on the complex.
Glucocorticoid action on protein synthesis and protein breakdown in isolated skeletal muscles
Authors: McGrath Josephine A. , Goldspink David F.
Published: August 2015

The direct actions of glucocorticoid hormones on protein turnover were studied in isolated soleus muscles. These steroids were found to decrease the rates of both protein synthesis and protein breakdown within 3 h and 4 h respectively. Synthetic steroids (e.g. dexamethasone) were found to be more potent than naturally secreted hormones (e.g. cortisol) in inducing these changes, but only at concentrations in vitro less than 10nm.
Enzymic synthesis of indol-3-ylacetyl-myo-inositol galactoside
Authors: Corcuera L J , Michalczuk L , Bandurski R S
Published: August 2015

Extracts of immature kernels of Zea mays catalysed the synthesis of indol-3-ylacetyl-myo-inositol galactoside from indol-3-ylacetyl-myo-inositol and UDP-galactose. Addition of 2-mercaptoethanol was required for stability of the catalytic activity during dialysis. The enzyme could be fractionated with (NH4)2SO4, and 55% of the activity was recovered in the 30-60%-saturation fraction. The product of the reaction contained radioactivity from UDP-[U-14C]galactose and was identified as indol-3-ylacetyl-myo-inositol galactoside by gas chromatography-mass spectrometry. Therefore a UDP-galactose:indol-3-ylacetyl-myo-inositol galactosyltransferase (indol-3-ylacetyl-myo-inositol galactoside synthase) is present in developing kernels of Zea mays. The description of this enzyme, together with the enzymes described in the accompanying paper [Michalczuk & Bandurski (1982) Biochem. J. 207, 273-281] for the synthesis of indol-3-ylacetylglucose and indol-3-ylacetyl-myo-inositol, now provides mechanisms for the biosynthesis of one-half of the low-molecular-weight esters of indol-3-ylacetic acid in Zea mays.
Role of the N-terminus of glutathione in the action of yeast glyoxalase I
Authors: Douglas K T , Al-Timari A , D'Silva C , Gohel D I
Published: August 2015

A number of S-substituted glutathiones and the corresponding N-substituted S-substituted analogues have been found to be linear competitive inhibitors of yeast glyoxalase I at 26 degrees C over the pH range 4.6-8.5. N-Acetylation of S-(p-bromobenzyl)glutathione weakens binding by 13.7-fold. N-benzoylation by 25.6-fold, N-trimethylacetylation by 53.3-fold and N-carbobenzoxylation by 7.8-fold, indicating a minor steric component in the binding at the N-site. The Ki-weakening effect of N-substitution of glutathione depends on the chemical nature of the S-substituent, indicating flexibility in the glutathione and/or glyoxalase I contributions to the binding site for glutathione derivatives. The effect of N-acylation on Ki is in accord with a charge interaction of the free enzyme with S-blocked glutathione in a region of reasonably high dielectric constant. There is a slight pH effect on Ki for S-(m-trifluoromethylbenzyl)glutathione but not for S-(p-bromobenzyl)glutathione.
Fluorescence and circular-dichroism properties of pig intestinal calcium-binding protein (Mr=9000), a protein with a single tyrosine residue
Authors: O'Neil J D , Dorrington K J , Kells D I C , Hofmann T
Published: August 2015

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.
Measurements of reducing end groups on bovine vitreous-humour hyaluronic acid by reaction with [14C]cyanide
Authors: Swann D A , Silver F H , Sotman S L , Hermann H
Published: August 2015

Bovine vitreous-humour sodium hyaluronate was purified by precipitation with cetylpyridinium chloride, CsCl-density-gradient sedimentation and gel-permeation chromatography. The number of reducing end groups in two similarly prepared hyaluronate samples was determined by reaction with K14CN, and measurements of intrinsic viscosity were performed to determine whether this reaction caused degradation of the hyaluronate. The intrinsic viscosity of one hyaluronate sample was 192ml/g, compared with a value of 187ml/g after reaction with [14C]cyanide, which indicates that the labelling reaction did not cause depolymerization of the hyaluronate. The Mr calculated from these viscosity values is approx. 60000. Fractionation of the [14C]cyanide-labelled hyaluronate by gel chromatography showed that it was composed of a polydisperse population of molecules with calculated chain lengths, based on the ratio of [14C]cyanide to uronic acid, ranging in molecular weight from 9000 to 264000, with an average Mr of 63200. On the basis of these measurements it is concluded that reaction with [14C]cyanide does not cause degradation of bovine vitreous-humour hyaluronate polysaccharide chains and that reaction with [14C]cyanide can be used to determine the molecular weight of this hyaluronate.
A quantitative study of the biospecific desorption of rat liver (M4) lactate dehydrogenase from 10-carboxydecylamino-Sepharose. Determination of the number of ligand-binding sites blocked on adsorption
Authors: Kyprianou P , Yon R J
Published: August 2015

1. The theory of Nichol, Ogston, Winzor & Sawyer [(1974) Biochem. J. 143, 435-443] for quantitative affinity chromatography, when adapted for use with a non-specific column from which a multi-site protein can be specifically desorbed by its free ligand, permits determination of the concentration of adsorption sites on the column, their adsorptive affinity (as an association constant) and either the intrinsic (site) constant for ligand-binding to the protein or an ‘occlusion coefficient’ (defined as the number of ligand-binding sites blocked on adsorption), one of which must be known. 2. The theory has been applied to the NADH-specific desorption of rat liver M4 lactate dehydrogenase from 10-carboxydecylamino-Sepharose. It suggests that most of the enzyme molecules are adsorbed with at least two NADH-binding sites blocked, indicating an extensive adsorption interface in relation to the protein surface. Other chromatographic parameters were also determined for the system. 3. Among topics discussed are (a) factors affecting the experimentally determined value for the number of blocked sites, (b) the nature of the adsorption sites on the column and (c) the similarity of the analysis to that for determining Hill coefficients, and other possible applications.
Haem disorder in reconstituted human haemoglobin
Authors: Docherty John C. , Brown Stanley B.
Published: August 2015

Degradation of the haem of haemoglobin (used as a chemical probe of the haem–protein relationship), suggests that reconstituted human haemoglobin contains significant haem disorder. This results from the insertion of haem into globin with an orientation 180° different from the natural orientation. Haem disorder also slowly occurs in methaemoglobin solutions.
Regulation of protein accumulation in cultured cells
Authors: Ballard F J
Published: August 2015

1. A technique is described whereby protein synthesis, protein breakdown and net protein accumulation are measured separately in monolayer cultures of mammalian cells. All rates are expressed as microgram of protein per 18 h incubation. 2. Under most incubation conditions with either L6 rat myoblasts or T47D human breast carcinoma cells the rates of protein accumulation, determined directly, agreed with the rates obtained by subtracting protein breakdown from protein synthesis. 3. Foetal calf serum, human and bovine colostrum, human milk and insulin increased protein accumulation in both cell lines, mainly as a consequence of effects on protein synthesis. 4. NH4Cl, in addition to inhibiting protein breakdown in both cell lines in the presence and in the absence of serum, stimulated protein synthesis in L6 myoblasts. 5. Leupeptin slightly inhibited protein breakdown without affecting protein-synthesis rates. 6. Cycloheximide almost completely inhibited protein synthesis, but restricted the net loss of cell proteins under most conditions because protein-breakdown rates were also decreased. 7. The assumptions, limitations and potential application of this technique for evaluating changes in protein turnover are described.
The development of γ-glutamyltransferase in a pig renal-epithelial-cell line in vitro. Relationship to amino acid transport
Authors: Sepúlveda F V , Burton K A , Pearson J D
Published: August 2015

The pig kidney-cell line, LLC-PK1, possesses gamma-glutamyltransferase with properties similar to those of the purified renal enzyme. gamma-Glutamyltransferase activity increases after cells are seeded at low density to reach values comparable with those found in kidney cortex when the cells are fully confluent. This increase is paralleled by an increase in alpha-methyl D-glucoside uptake. In contrast, the uptakes of L-leucine and L-alanine decrease during this time. These results suggest that gamma-glutamyltransferase is not important as a mediator of the renal transport of neutral amino acids.
Calcium ion fluxes induced by the action of α-adrenergic agonists in perfused rat liver
Authors: Reinhart P H , Taylor W M , Bygrave F L
Published: August 2015

Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.
Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes
Authors: Chander Avinash , Fisher Aron B. , Strauss Jerome F.
Published: August 2015

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-3H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-3H]palmitic acid, 458±18pmol of [1-14C]oleic acid and 252±15pmol of [U-14C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [3H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-14C]glucose increased by 125%. Incorporation of [1-14C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.
Parotid microsomal Ca2+ transport. Subcellular localization and characterization
Authors: Kanagasuntheram P , Teo T S
Published: August 2015

Rat parotid gland homogenates were fractionated into mitochondrial, heavy microsomal and light microsomal fractions by differential centrifugation. ATP-dependent 45Ca2+ uptake by the subcellular fractions paralleled the distribution of NADPH-cytochrome c reductase, an enzyme associated with the endoplasmic reticulum. The highest rate of Ca2+ uptake was found in the heavy microsomal fraction. Ca2+ uptake by this fraction was dependent on the presence of ATP and was sustained at a linear rate by 5 mM-oxalate. Inhibitors of mitochondrial Ca2+ transport had no effect on the rate of Ca2+ uptake. Na+ and K+ stimulated Ca2+ uptake. At optimal concentrations. Na+ stimulated Ca2+ uptake by 120% and K+ stimulated Ca2+ uptake by 260%. Decreasing the pH from 7.4 to 6.8 had little effect on Ca2+ uptake. The Km for Ca2+ uptake was 3.7 microM free Ca2+ and 0.19 mM-ATP. Vanadate inhibited Ca2+ uptake; 60 microM-vanadate inhibited the rate of Ca2+ accumulation by 50%. It is concluded that the ATP-dependent Ca2+ transport system is located on the endoplasmic reticulum and may play a role in maintaining intracellular levels of free Ca2+ within a narrow range of concentration.
Kinetic studies on cytochrome c oxidase inserted into liposomal vesicles. Effect of ionophores
Authors: Sarti P , Colosimo A , Brunori M , Wilson M T , Antonini E
Published: August 2015

Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the ‘pulsed’ form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.
Ligand binding, conformational change and plasma elimination of human, mouse and rat α-macroglobulin proteinase inhibitors
Authors: Gonias S L , Balber A E , Hubbard W J , Pizzo S V
Published: August 2015

Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a ‘slow’ to ‘fast’ electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.
Heterogeneity of rat goblet-cell mucin before and after reduction
Authors: Fahim R E F , Forstner G G , Forstner J F
Published: August 2015

Goblet-cell mucin of rat small intestine was purified from mucosal scrapings by using centrifugation, Sepharose 4B and Sepharose 2B chromatography. The mucin was applied in low concentrations (1 microgram/track) to slab gels containing 0.5% agarose/2% (w/v) polyacrylamide, and bands were detected after electrophoresis by silver stain or by fluorography of 3H-labelled mucin. Before reduction the mucin contained three distinct components: a polymeric species at the top of the gel and two large glycoproteins of higher mobility. After reduction, the polymer disappeared, the two glycoproteins remained unchanged, and two glycopeptide bands of higher mobility appeared. In addition, a non-glycosylated, heavily stained peptide of mol.wt. 118000 was detected. The individual mucin components were partially separated on Sepharose 2B, 0.2M-NaCl/1% sodium dodecyl sulphate being used as eluant. Individual amino acid and carbohydrate analyses suggested that the glycosylated components, despite their differences in size, had identical profiles. The 118000-mol.wt. peptide had a very different amino acid profile, with much less serine, threonine and proline. Glycine and aspartic and glutamic acids comprised 34% of the total amino acids. Thus the ‘native’ mucin is a heterogeneous structure containing at least two non-covalently associated glycoproteins plus polymeric material. The latter is stabilized by disulphide bonds and consists of several glycopeptides of different size as well as a ‘link’ peptide of mol.wt. 118000.
Phosphorylation of cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T) by cardiac phospholipid-sensitive Ca2+-dependent protein kinase
Authors: Katoh N , Wise B C , Kuo J F
Published: August 2015

Cardiac phospholipid-sensitive Ca2+-dependent protein kinase phosphorylated cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T), present either as the free form or as the troponin-tropomyosin complex. Exhaustive phosphorylation of troponin I and of troponin T revealed that 1.7 and 2 mol of phosphate was incorporated/mol of the subunits respectively. Cyclic AMP-dependent protein kinase, though incorporating 0.8 mol of phosphate/mol of troponin I, was unable to phosphorylate troponin T. Phosphorylation of troponin I (apparent Km = 3.4 microM; Vmax. = 2.6 mumol/min per mg of enzyme) or troponin T (apparent Km = 0.3 microM; Vmax. = 0.5 mumol/min per mg of enzyme) by the Ca2+-dependent enzyme was inhibited by various agents, such as adriamycin, palmitoylcarnitine, trifluoperazine, melittin and N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide (compound W-7). Ca2+ antagonists (such as verapamil), forskolin and ouabain were ineffective. These findings indicate that troponin I and troponin T were effective substrates for this species of Ca2+-dependent protein kinase, suggesting its potential regulatory role in the contractile activity of myofibrils modulated by troponin.
Gel-permeation high-performance liquid chromatography as a powerful technique for rapid analysis of calcitriol (1,25-dihydroxycholecalciferol) receptors
Authors: Danan J L , Mathieu H
Published: August 2015

We used gel-permeation high-performance liquid chromatography to detect cytosolic and nuclear calcitriol (1,25-dihydroxycholecalciferol) receptors. This new convenient technique allowed accurate separation of the calcitriol receptor from other vitamin D-binding proteins. Compared with sucrose-density-gradient centrifugation, it has several advantages including, in particular, its rapidity.
Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing
Authors: Jones C S , Shankaran P , Davidson D J , Poulos A , Callahan J W
Published: August 2015

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.
Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro
Authors: Saklatvala J , Sarsfield S J , Pilsworth L M C
Published: August 2015

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.
The regulatory properties of rabbit muscle pyruvate kinase. The influence of substrate concentrations
Authors: Ainsworth S , Kinderlerer J , Gregory R B
Published: August 2015

The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.
Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing
Authors: Middle F A , Bannister A , Bellingham A J , Dean P D G
Published: August 2015

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.
The alkaline phospholipase A1 of rat liver cytosol
Authors: Dawson R M C , Irvine R F , Hemington N L , Hirasawa K
Published: August 2015

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.
The relationship between placental protein synthesis and transfer of amino acids
Authors: Carroll M J , Young M
Published: August 2015

The relationship between placental protein synthesis and transfer of amino acids from mother to foetus was studied in the guinea pig, by using [U-14C]-lysine, -leucine, -glycine, -aspartate and -alpha-aminoisobutyrate. The uptake of label by protein was 12-16% of total label transferred. Cycloheximide inhibited incorporation of all naturally occurring amino acids into protein by 81-96% and transfer by 62-75%; the concentration of label in the free pool was increased for each. These findings were confirmed when specific-radioactivity measurements were made with L-[U-14C]lysine. The transfer of the non-protein amino acid alpha-aminoisobutyrate was not significantly decreased by cycloheximide. A model, linking protein synthesis to the generation of a transfer pool of amino acids, is proposed whereby inhibition of protein synthesis decreases the amount of amino acid available for transfer.
Isolation of mannose-binding proteins from human and rat liver
Authors: Wild J , Robinson D , Winchester B
Published: August 2015

A binding assay for the detection of mannose-binding proteins was developed, which uses a ligand of mammalian origin, 125I-labelled bovine pancreatic ribonuclease B. The binding assay was validated by using the recognized mannose-binding protein, concanavalin A. Microgram quantities of concanavalin A or mannose-binding proteins could be assayed. A mannose-binding protein was isolated from rat liver by affinity chromatography on mannose-Sepharose 6B. It has a Mr of approx. 900000 under non-dissociating conditions and contains a subunit of approx. 34000 Mr. When ribonuclease B-Sepharose was used as a ligand for affinity chromatography, the predominant mannose-binding material isolated from rat liver had a native Mr of 205000-225000 and consisted largely of a subunit of Mr 70000, which yielded subunits of Mr 28500 and 34000 on reduction. It is suggested that different mannose-binding proteins are isolated by the two affinity-chromatography ligands. A mannose-binding protein was also purified from human liver by affinity chromatography on mannose-Sepharose 6B. It has a native Mr of over 1000000 and consists of subunits with Mr 28000 and 30500. Its isolation suggests that mannose-mediated endocytosis or intracellular transport of glycoproteins occurs in human liver.
Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1
Authors: Moss E J , Judah D J , Przybylski M , Neal G E
Published: August 2015

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that not only facilitates improved resolution of the compound but that, by manipulation of the pH, is also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, has been used in 1H-n.m.r. and mass-spectroscopic studies. Results were obtained that support the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a gamma-glutamyltransferase-catalysed-transferable glutamate moiety. These data are consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1.
Effects of anabolic agents on protein breakdown in L6 myoblasts
Authors: Ballard F J , Francis G L
Published: August 2015

1. Protein degradation in rat L6 myoblasts is inhibited by high concentrations of insulin as well as by foetal bovine serum and bovine colostrum, mixtures rich in growth-factor activity. 2. Growth factors achieve maximal effects within 2 h after addition to the cell cultures, but these diminish with time. Indeed, during incubations greater than 12 h, foetal calf serum actually stimulates protein breakdown. The changed response, however, is not due to the depletion of growth factors from serum. 3. Protein breakdown is stimulated by dexamethasone by a process that takes several hours to be expressed, but is more pronounced over a 4 h measurement period than over 18h. The glucocorticoid response is prevented by insulin or by cycloheximide. 4. Anabolic agents such as trenbolone, diethylstilboestrol and testosterone do not alter rates of intracellular protein breakdown and do not interfere with the glucocorticoid-induced catabolic response. 5. The results are consistent with anabolic steroids and related agents acting indirectly on muscle, perhaps via altering concentrations of growth factors of the somatomedin type.
Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum
Authors: Raeymaekers L , Wuytack F , Eggermont J , De Schutter G , Casteels R
Published: August 2015

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.
Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction
Authors: Dawson A P , Fulton D V
Published: August 2015

1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.
Control of activation of liver RNA polymerase I occurring after re-feeding of protein-depleted mice
Authors: Haim L , Iapalucci-Espinoza S , Conde R , Franze-Fernández M T
Published: August 2015

Shortly after feeding protein-depleted mice with a meal containing protein, the RNA polymerase I activity in isolated liver nuclei shows a 2-fold increase over the values in the nuclei of either normal or protein-depleted mice. The activity of the RNA polymerase I solubilized from nuclei of re-fed mice was slightly enhanced, probably reflecting an increase in enzyme amount. However, this increase only accounts for about 30% of the stimulation of transcription in the intact nuclei. Administration of pactamycin, an inhibitor of protein synthesis, to normal or protein-depleted mice has almost no inhibitory effect on the RNA polymerase I activity in the isolated nuclei. On the contrary, within 15 min after treatment with the drug, the stimulated activity in nuclei from re-fed mice declines towards the values in normal or protein-depleted mice and then remains constant. The activity of the solubilized enzyme remains slightly elevated for at least 2 1/2 h after re-fed mice are treated with pactamycin. These observations indicate that the stimulation of the RNA polymerase I activity in the intact nuclei after re-feeding is controlled by mechanisms other than an increase in the enzyme amount and suggest the presence of short-lived proteins required for inducing an activated state of transcription.
The role of 5-aminolaevulinate synthase, haem oxygenase and ligand formation in the mechanism of maintenance of cytochrome P-450 concentration in hepatocyte culture
Authors: Hockin L J , Paine A J
Published: August 2015

The present work shows that the ability of pyridines e.g. metyrapone, to maintain the cytochrome P-450 concentration in cultured hepatocytes is not due to their ability to alter the 5-aminolaevulinate synthase and haem oxygenase activities of the hepatocytes. Since ligands such as metyrapone will prevent the cobalt-mediated loss of hepatic cytochrome P-450 in rats, the hypothesis that ligand formation is the mechanism of maintenance of the cytochrome in hepatocyte culture was tested. The observation that non-pyridine ligands will maintain the cytochrome P-450 concentration supports this hypothesis.
Modulation of active Ca2+ uptake by the islet-cell endoplasmic reticulum
Authors: Colca J R , Kotagal N , Lacy P E , McDaniel M L
Published: August 2015

The possible effects of calmodulin and cyclic AMP on active Ca2+ uptake by the islet-cell endoplasmic reticulum were investigated. Neither calmodulin nor cyclic AMP affected the rate of active Ca2+ uptake, or the steady-state filling capacity of the endoplasmic reticulum when measured in the absence of oxalate. Consistent with these results, calmodulin did not activate the Ca2+-stimulated ATPase activity associated with this cell fraction. During the course of these experiments., it was unexpectedly discovered that the rate of Ca2+ uptake, as well as the steady-state Ca2+ filling capacity of the endoplasmic reticulum, were markedly increased by unidentified factor(s) in the cytosol. This effect could be demonstrated by reconstitution of the membranes in cytosol, or by direct addition of fresh or dialysed cytosol to the Ca2+ uptake assays. The degree of activation by the cytosol indicates that the endoplasmic reticulum may play a prominent role in controlling β-cell Ca2+ concentrations and that the unidentified activator(s) present in the cytosol may be involved in regulation of this function.
Novel prostaglandin dehydrogenase in rat skin
Authors: Fincham N , Camp R
Published: August 2015

Present evidence suggests that skin is an important organ of prostaglandin metabolism. To clarify its role, the basic kinetics of 15-hydroxyprostaglandin dehydrogenase (PGDH) from rat skin were investigated with either NAD+ of NADP+ as co-substrate. Prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) were used as substrates and preliminary studies were made of the inhibitory effects of the reduced co-substrates NADH and NADPH. A radiochemical assay was used in which [3H]PGF2 alpha or [14C]PGE2 were incubated with high-speed supernatant of rat skin homogenates. The substrate and products were then extracted by solvent partition, separated by t.l.c. and quantified by liquid-scintillation counting. At linear reaction rates and at an NAD+ concentration of 10 mM the mean apparent Km for PGF2 alpha was 24 microM with a mean apparent Vmax. of 9.8 nmol/s per litre of reaction mixture. For PGE2 the mean apparent Km was 8 microM, with a mean apparent Vmax, of 2.7 nmol/s per litre of reaction mixture. With NADP+ as a co-substrate at a concentration of 5 mM a mean apparent Km of 23 microM was obtained for PGF2 alpha with a mean apparent Vmax. of 5.2 nmol/s per litre. For PGE2 values of 7.5 microM and 3.0 nmol/s per litre were obtained respectively. These results show that skin contains NAD+- and NADP+-dependent PGDH. An important finding was that the NADP+-linked enzyme gave Km values for PGE2 that were considerably lower than those reported for NADP+-linked PGDH from other tissues. Furthermore, preliminary inhibition studies with the NAD+-linked PGDH system indicate that this enzyme is not only inhibited by NADH, but also by NADPH, a property not previously reported for NAD+-linked PGDH derived from other tissues.