bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical synthesis and papain-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide
Authors: Mackenzie N E , Malthouse J P G , Scott A I
Published: August 2015

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.
The effect on photohaemolysis of variation in the structure of the porphyrin photosensitizer
Authors: de Paolis A , Chandra S , Charalambides A A , Bonnett R , Magnus I A
Published: August 2015

A comparison of the photosensitizing ability of a variety of porphyrins for photohaemolysis gives the following order of activity: protoporphyrin greater than deuteroporphyrin, mesoporphyrin, haematoporphyrin dimethyl ester much greater than haematoporphyrin diacetate, haematoporphyrin greater than haematoporphyrin monoacetate, coproporphyrin III, haematoporphyrin derivative, coproporphyrin III tetramethyl ester greater than uroporphyrin I, meso-tetra-(N-methyl-4-pyridinium)porphyrin tetratoluene-p-sulphonate, meso-tetra-(p-carboxyphenyl)porphyrin, protoporphyrin dimethyl ester, meso-tetra-(p-hydroxy-sulphonylphenyl)porphyrin tetrasodium salt, uroporphyrin III, deuteroporphyrin-3,8-disulphonic acid and protohaemin. The results for the metal-free porphyrins are rationalized in terms of solubility and partition properties, and a model is proposed for the incorporation of amphipathic porphyrins into the membrane lipid bilayer. Experiments with erythrocytes from patients with erythropoeitic protoporphyria and with normal erythrocytes to which porphyrin was added in a deuterium oxide medium do not lead to an increase in the rate of photohaemolysis. A possible explanation for this somewhat surprising observation is outlined.
Effect of ouabain on amino acid uptake by mouse ascites-tumour cells in the presence of nigericin
Authors: Johnson E , Eddy A A
Published: August 2015

Mouse ascites-tumour cells oxidizing lactate, in a modified Ringer solution, concentrated 2-aminoisobutyrate, L-methionine or 2-(methylamino)isobutyrate about 20-fold from a 0.4 mM solution in the presence of 2-3 micrograms of nigericin/mg cellular dry wt. The ionophore increased cellular [Na+] to almost 100 mM when extracellular [Na+] was about 45 mM. Either valinomycin or the two mitochondrial inhibitors oligomycin and antimycin acting together each markedly lowered the extent to which the tumour cells concentrated amino acid, from the above factor of about 20 to roughly 2-fold. Ouabain (1 mM) had a similar effect, and further raised cellular [Na+]. The sodium pump appeared to be closely involved in amino acid uptake under these conditions.
Characterization of protein kinase C and its role in catecholamine secretion from bovine adrenal-medullary cells
Authors: Brocklehurst K W , Morita K , Pollard H B
Published: August 2015

Protein kinase C activity towards exogenous histone was detected in a cytosolic fraction of bovine adrenal medulla. The enzyme was dependent on Ca2+ and phosphatidylserine for its activity, with half-maximal activation being achieved at approx. 18 microM free Ca2+ and 8 micrograms of phosphatidylserine/ml. Both diolein and 4 beta-phorbol 12-myristate 13-acetate (TPA) decreased the Ca2+ requirement of the enzyme, half-maximal activation being obtained at approx. 12 microM and 9 microM free Ca2+ respectively in the presence of these agents. Many endogenous proteins in the adrenal-medullary cytosolic fraction were detected whose phosphorylation was dependent on the presence of both Ca2+ and phosphatidylserine. TPA stimulated catecholamine release from cultured bovine adrenal-chromaffin cells in a Ca2+-dependent manner. A23187 also stimulated catecholamine secretion, and at sub-optimal concentrations of TPA and A23187 a synergistic secretory response was obtained. These results are consistent with protein kinase C having a regulatory role in exocytosis in bovine adrenal chromaffin cells.
Interactions of soya-bean agglutinin with purified glycoconjugates and soya-bean seed components
Authors: Bond H M , Chaplin M F , Bowles D J
Published: August 2015

A radioaffinity assay for lectin binding to receptors was developed and characterized by using the interactions between soya-bean agglutinin and four glycoconjugates, namely thyroglobulin, galactomannan, fetuin and asialofetuin. On application of the assay to soya-bean extracts a wide range of seed components were found to have the capacity to interact with soya-bean agglutinin. These included both trichloroacetic acid-soluble and trichloroacetic acid-insoluble glycoconjugates and two classes of particulate matter distinguished by their differential solubility in Triton X-100.
Butyrate formation from glucose by the rumen protozoon Dasytricha ruminantium
Authors: Yarlett N , Lloyd D , Williams A G
Published: August 2015

Production of butyrate by the holotrich protozoon Dasytricha ruminantium involves the enzymes of glycolysis, pyruvate:ferredoxin oxidoreductase, acetyl-CoA:acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxyacyl-CoA hydro-lyase, 3-hydroxyacyl-CoA reductase, phosphate butyryltransferase and butyrate kinase. Subcellular fractionation by differential and density-gradient centrifugation on sucrose gradients indicated that all those enzymes except pyruvate:ferredoxin oxidoreductase were non-sedimentable at 6 × 10(6) g-min. Butyrate kinase and phosphate butyryltransferase were associated with the large- and small-granule fractions. Thus, although metabolic reactions necessary for butyrate production proceed predominantly in the cytosol, hydrogenosomes play a key role in the conversion of pyruvate into acetyl-CoA.
The energetics of early platelet responses. Energy consumption during shape change and aggregation with special reference to protein phosphorylation and the polyphosphoinositide cycle
Authors: Verhoeven A J M , Gorter G , Mommersteeg M E , Akkerman J W
Published: August 2015

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.
Sidedness studies of thylakoid phosphatidylglycerol in higher plants
Authors: Unitt M D , Harwood J L
Published: August 2015

The transmembrane distribution of phosphatidylglycerol was determined in thylakoids from barley (Hordeum vulgare), lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. Phospholipase A2 and phospholipase D digestion and chemical-labelling methods were used. Phosphatidylglycerol was preferentially localized in the outer (stromal) leaflet. The proportion of the phospholipid in this leaflet ranged from about 66% in pea to about 75% for barley and lettuce thylakoids. One of the main fatty acids, trans-delta 3-hexadecenoic acid, was exclusively located in the outer leaflet in all three plant types. The data are discussed in relation to suggested roles for phosphatidylglycerol in thylakoid function.
Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans
Authors: Morgan N G , Rumford G M , Montague W
Published: August 2015

Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.
Thromboplastin (tissue factor) in plasma membranes of human monocytes
Authors: Hetland O , Brovold A B , Holme R , Gaudernack G , Prydz H
Published: August 2015

The synthesis of thromboplastin, a potent trigger of blood coagulation, can be induced in human peripheral blood monocytes. Indirect evidence suggests that newly synthesized thromboplastin becomes in part available on the cell surface. We have attempted to study the localization and availability of thromboplastin more directly by isolating plasma membranes from isolated human peripheral blood monocytes. The specific activities of the plasma membrane markers increased 16-22-fold in these preparations with a recovery of about 15%. The contamination by mitochondria, lysosomes, nuclei and endoplasmic reticulum was low as estimated by marker enzymes and electron microscopy. In both unstimulated and stimulated monocytes thromboplastin was largely recovered in this plasma membrane fraction, providing direct evidence for its membrane localization. Phospholipase C (E.C. 3.1.4.3) is a potent inactivator of thromboplastin through its hydrolysis of the phospholipids necessary for thromboplastin activity [Otnaess, Prydz, Bjørklid & Berre (1972) Eur. J. Biochem. 27, 238-243]. About 70% of the total membrane thromboplastin activity was inactivated when whole cells were treated with phospholipase C and the membranes subsequently isolated. Following stimulation to induce thromboplastin synthesis, the plasma membranes showed a shift in their relative content of phosphatidylcholine and phosphatidylethanolamine consistent with a transmethylation process.
Iodinated fibroblast β-glucuronidase as a ligand for receptor-mediated endocytosis
Authors: Dean M F , Diment S , Ostlünd C , Jenne B M , Contractor S
Published: August 2015

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 × 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 × 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.
Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys
Authors: Hedeager-Sørensen S , Kenny A J
Published: August 2015

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at −20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.
Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts
Authors: Hunt L A , Wright S E
Published: August 2015

In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of ‘truncated’ precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.
The control by ΔµH+ of the tonoplast-bound H+-translocating adenosine triphosphatase from rubber-tree (Hevea brasiliensis) latex
Authors: Marin B P
Published: August 2015

The relationship between tonoplast-bound ATPase activity and the magnitude of the electrochemical proton gradient has been investigated on tightly sealed vesicles prepared from rubber-tree (Hevea brasiliensis) latex. A variety of methods have been used to modify, either alone or together, the two components of the electrochemical proton gradient (delta mu H+). When the delta pH component was decreased either by titration with (NH4)2SO4 or by addition of protonophores or nigericin in the presence of K+, ATPase activity was stimulated. On the other hand, when the delta psi component was decreased either by addition of lipophilic cations or by addition of valinomycin in the presence of K+, ATPase activity decreased. It is concluded that activity of the tonoplast-bound ATPase is regulated by changes in the electrochemical proton gradient across the tonoplast, so that, once the maximum proton gradient is established across the tonoplast, any perturbation of the equilibrium state should result in the increased rate of ATP hydrolysis as the enzyme attempts to re-establish the initial gradient.
Isolation and partial characterization of canine epidermal growth factor/urogastrone
Authors: Kobayashi R , Reeve J R , Walsh J H
Published: August 2015

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.
The NADH oxidase system (external) of muscle mitochondria and its role in the oxidation of cytoplasmic NADH
Authors: Rasmussen U F , Rasmussen H N
Published: August 2015

An exo-NADH oxidase system [NADH oxidase system (external)], effecting intact-mitochondrial oxidation of added NADH, was studied in pigeon heart mitochondria. Breast muscle mitochondria showed an equal specific activity of the system. The exo-NADH oxidase activity (200 micron mol of NADH/min per g of protein) equalled two-thirds of the State-3 respiratory activity with malate + pyruvate or one-seventh of the total NADH oxidase activity of heart mitochondria. The activity was not caused by use of proteinase in the preparation procedure and all measured parameters were very reproducible from preparation to preparation. The activity is therefore most likely not due to preparation artefacts. The exo-NADH oxidase system is present in all mitochondria in the preparation and is not confined to a subpopulation. The system reduced all cytochrome anaerobically and direct interaction with all cytochrome oxidase was demonstrated by interdependent cyanide inhibition. The exo-NADH oxidase system seems to be located at the outer surface of the mitochondrial inner membrane because, for instance, only this system was rapidly inhibited by rotenone, and ferricyanide could act as acceptor in the rotenone-inhibited system (reductase activity = 20 times oxidase activity). In the presence of antimycin, added NADH reduced only a part of the b-cytochromes. Freezing and thawing the mitochondria, one of the methods used for making them permeable to NADH, destroyed this functional compartmentation. The characteristics of the exo-NADH oxidase system and the malate-aspartate shuttle are compared and the evidence for the shuttle's function in heart in vivo is re-evaluated. It is proposed that oxidation of cytoplasmic NADH in red muscles primarily is effected by the exo-NADH oxidase system.
Transcription of Rhodospirillum rubrum atp operon
Authors: Falk G , Walker J E
Published: August 2015

The photosynthetic non-sulphur bacterium Rhodospirillum rubrum contains a cluster of five genes encoding the subunits of F1-ATPase [Falk, Hampe & Walker (1985) Biochem. J. 228, 391-407]. Transcription of these genes has been studied by two methods, transcriptional mapping with S1 nuclease and primer extension analysis. Thereby a 5'-end in RNA derived from this region has been demonstrated at a guanine residue 236 bases before the initiation codon of the gene for the delta-subunit, the first in this cluster. DNA sequences on the 5' side of this nucleotide show some similarity to promoters in Escherichia coli, but are not apparently related to sequences upstream of the Rhodopseudomonas blastica atp operon. A 3'-end in RNA derived from this gene cluster has been demonstrated by S1-nuclease mapping. This is found before a run of thymidylate residues in the DNA, on the 3' side of a region of dyad symmetry. In E. coli these features are characteristic of rho-independent transcriptional termination signals. It appears from these studies and from the organization of the genes that the five genes in the atp cluster may be co-transcribed from this promoter and that transcripts terminate at the region of dyad symmetry.
Major changes in phosphorylation of chromatin-associated non-histone proteins accompany development in the slime mould Dictyostelium discoideum
Authors: Sinclair J H , Rickwood D
Published: August 2015

Striking changes in protein and RNA synthesis were shown to accompany development in the slime mould Dictyostelium discoideum. These changes are, at least in part, directly attributable to control at the level of transcription. Analysis of nuclear proteins and their states of phosphorylation by two-dimensional gel electrophoresis and autoradiography showed only minor changes in the species of proteins detectable by Coomassie Blue staining during the vegetative growth and development phases. Major changes, however, were detected in their patterns of phosphorylation, with major differences from the vegetative pattern occurring during both early development (0-2h) and late development (8-12h). These changes coincide with major changes in polypeptide synthesis and in nuclear and cytoplasmic RNA complexity.
The production and molecular properties of the zinc β-lactamase of Pseudomonas maltophilia IID 1275
Authors: Bicknell R , Emanuel E L , Gagnon J , Waley S G
Published: August 2015

The production and purification of a tetrameric zinc beta-lactamase from Pseudomonas maltophilia IID 1275 were greatly improved. Three charge variants were isolated by chromatofocusing. The subunits each contain two atomic proportions of zinc and (in two of the variants) one residue of cysteine. The thiol group is not required for activity, nor does it appear to bind to the metal. Replacement of zinc by cobalt, cadmium or nickel takes place at a measurable rate, and gives enzymes that are less active than the zinc enzyme. The properties of this enzyme differ from those of the other known zinc beta-lactamase, beta-lactamase II from Bacillus cereus. The amino acid sequence of the N-terminal 32 residues was determined; there is no similarity to the N-terminal sequences of other beta-lactamases.
Interactions with glutathione S-transferases of porphyrins used in photodynamic therapy and naturally occurring porphyrins
Authors: Smith A , Nuiry I , Awasthi Y C
Published: August 2015

Several naturally occurring porphyrins and porphyrins used in photodynamic therapy inhibit glutathione S-transferase isoenzymes either purified from rat liver or lung or in cytosol from normal and from cancerous (Morris 7288C hepatoma) liver. Although differences occur in the type and amount of transferases in normal and cancerous liver and in the liver of rats bearing an extrahepatic tumour, these enzymes are potential binding sites for porphyrins. Porphyrin structure is an important factor in determining the affinity of binding, as shown by the relative inhibitory effectiveness. Of the dicarboxylic porphyrins in the mixture used clinically, OO'-diacetylhaematoporphyrin and monohydroxyethylmonovinyldeuteroporphyrin are more effective inhibitors than haematoporphyrin and protoporphyrin IX. Of the naturally occurring porphyrins the order of effectiveness is protoporphyrin IX (dicarboxylic) greater than coproporphyrin (tetracarboxylic) greater than uroporphyrin (octacarboxylic) and type I greater than type III isomers of both uroporphyrin and coproporphyrin, and the synthetic tetra-meso-phenylporphinetetrasulphonate is a better inhibitor (apparent Ki = 250 nM) than coproporphyrin, which contains a comparable number of negative charges. In addition, iron-porphyrin chelates are more effective inhibitors of the transferases, with 25-fold decrease in Ki value, than the free porphyrins. These results indicate that one means whereby porphyrins accumulate in tissues is the occupation of intracellular binding sites, such as the transferases. Since porphyrins inhibit the activity of these important detoxifying enzymes, there will be metabolic consequences to the cell.
Interacting effects of l-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase
Authors: Bird M I , Saggerson E D
Published: August 2015

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.
Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases
Authors: Goueli S A , Davis A T , Hiipakka R A , Liao S , Ahmed K
Published: August 2015

Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases.
Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent
Authors: Helman J , Roth G S , Baum B J
Published: August 2015

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.
Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3
Authors: Hellman U , Eggertsen G , Engström A , Sjöquist J
Published: August 2015

Human C3d (try-C3d), prepared from trypsin-digested C3, was fragmented by cleavage with CNBr. Eight peptides were defined and separated by h.p.l.c. on reversed-phase columns. By automatic Edman degradation the complete sequences of five peptides and partial sequences of three peptides were determined. To obtain overlapping peptides the latter three fragments were digested with trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase, after which the fragments were separated on reversed-phase columns. Two of the CNBr-cleavage peptides were completely sequenced, and 70% of the sequence of the remaining CNBr-cleavage peptide was determined. The non-sequenced part represents a very hydrophobic segment of try-C3d. The sequence data obtained represent 90% of the primary structure of try-C3d. Alignment of the CNBr-cleavage fragments was made easier by comparison with the cDNA sequence of mouse pro-C3 [Wetsel, Lundwall, Davidson, Gibson, Tack & Fey (1984) J. Biol. Chem. 259, 13857-13862]. Comparison of try-C3d with the equivalent part of human C4B revealed an extensive sequence homology in the N-terminal half of the molecules.
Altered release of carnitine palmitoyltransferase activity by digitonin from liver mitochondria of rats in different physiological states
Authors: Zammit V A , Corstorphine C G
Published: August 2015

The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.
Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo
Authors: Ayad S , Chambers C A , Shuttleworth C A , Grant M E
Published: August 2015

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide ‘maps’, were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].
Ribitol dehydrogenase of Klebsiella aerogenes. Sequence of the structural gene
Authors: Loviny T , Norton P M , Hartley B S
Published: August 2015

The ribitol dehydrogenase gene was cloned from wild-type Klebsiella aerogenes and also from a transducing phage lambda prbt which expresses the rbt operon constitutively. The coding sequence for 249 amino acids is separated from the following D-ribulokinase gene by 31 base pairs containing three stop codons, one of which overlaps the ribosome binding site for D-ribulokinase. Three residues in the amino acid sequence differ from that predicted from the DNA sequence: Asp-212 for Asn-212 is probably a protein sequencing error, but -Ala-Val- for -Ser-Ser- at 146-147 appears to be a ‘neutral mutation’ that may have arisen during prolonged chemostat selection of a strain that superproduces the enzyme from which the protein sequence was determined.
Partial characterization of lysyl oxidase from several human tissues
Authors: Kuivaniemi H
Published: August 2015

Lysyl oxidase activity was assayed in urea extracts of a number of human tissues, proving to be highest in skin. Antibodies to human placental lysyl oxidase completely inhibited the activity of crude lysyl oxidase from all the human tissues studied, with no significant differences in the amounts of antiserum required for 50% inhibition. By contrast, marked differences were found in this value between skin tissue samples from different species. The Mr of lysyl oxidase in crude extracts of human skin and in the medium of cultured human skin fibroblasts was 30 000 by gel filtration, no active species with a higher Mr being detectable. Four forms of lysyl oxidase activity were seen in DEAE-cellulose chromatography of urea extract from human skin, all having Mr 30 000. Antibodies to human placental lysyl oxidase stained a 30 000-Mr protein in urea extracts of all the human tissues studied and in the medium of cultured human skin fibroblasts when examined by immunoblotting after sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, but they also stained high-Mr material. The findings suggest that there are no immunologically distinct lysyl oxidase isoenzymes in the various human tissues and that the true Mr of lysyl oxidase in crude urea extracts is 30 000.
Hormone-induced actin polymerization in rat hepatoma cells and human leucocytes
Authors: Rao K M K , Betschart J M , Virji M A
Published: August 2015

Treatment of rat hepatoma cells with insulin, glucagon, thyroxine (T4) and triiodothyronine (T3) caused a concentration-dependent decrease in the monomeric actin content as measured by the deoxyribonuclease-I inhibition assay. Similarly, human peripheral blood neutrophils responded with a decrease in monomeric actin content when stimulated with T4, T3 and the adrenergic agonists phenylephrine and isoprenaline. The effect of phenylephrine could be blocked by phentolamine, demonstrating the specificity of the interaction. These observations suggest that hormone-induced actin changes might be an important event in response to both cell-surface-reactive hormones, such as insulin, glucagon and adrenergic agents, and those hormones that act through intracellular receptors, such as thyroid hormones. It is suggested that changes in actin state may have a role in metabolic regulation and cell growth.
Purification, immunological and biochemical characterization of rat 28 kDa cholecalcin (cholecalciferol-induced calcium-binding proteins). Identity between renal and cerebellar cholecalcins
Authors: Intrator S , Elion J , Thomasset M , Brehier A
Published: August 2015

The cholecalcins are intracellular vitamin D-dependent calcium-binding proteins. High concentrations of 28 kDa cholecalcins have been found in the kidneys and cerebella of birds and mammals. However, whereas the synthesis of the renal protein is vitamin D-dependent, that of the cholecalcin in the cerebella of young growing chicks and rats is apparently not. In the present study a range of immunological, physicochemical and structural characteristics of renal and cerebellar cholecalcins isolated from a single species, the rat, has been examined to ascertain what, if any, differences there are between them, other than the vitamin D-dependence. Both proteins behaved in exactly the same way during purification and showed complete immunocross-reactivity in Ouchterlony double immunodiffusion and radioimmunoassay. No difference could be detected between them on electrophoresis under denaturing conditions or on two-dimensional isoelectric focusing/electrophoresis. Their amino acid compositions were very similar, as were their u.v.-absorption spectra and their chymotryptic and tryptic peptide maps. The N-terminal sequence was found to be Gly-Gly-Val-Ser... for both cholecalcins. We have thus been unable to show any significant difference between the two proteins and suggest that the 28 kDa cholecalcins of the rat cerebellum and kidney are extremely similar, if not identical.
Effects of bis(guanylhydrazones) on the activity and expression of ornithine decarboxylase
Authors: Nikula P , Alhonen-Hongisto L , Jänne J
Published: August 2015

Derivatives of glyoxal bis(guanylhydrazone) (GBG), such as methylglyoxal bis(guanylhydrazone) and ethylglyoxal bis(guanylhydrazone), are potent inhibitors of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the key enzyme required for the synthesis of spermidine and spermine. These compounds, but not the parent compound, induce a massive accumulation of putrescine, partly by blocking the conversion of putrescine into spermidine, but also by strikingly stimulating ornithine decarboxylase (ODC; EC 4.1.1.17) activity. The mechanism of the stimulation of ODC activity and enhanced accumulation of the enzyme protein apparently involved a distinct stabilization of the enzyme against intracellular degradation. However, although the parent compound GBG also stabilized ODC, it powerfully inhibited the enzyme activity and the accumulation of immunoreactive protein in cultured L1210 leukaemia cells. Kinetic considerations indicated that, in addition to the stabilization, all three compounds, GBG in particular, inhibited the expression of ODC. It is unlikely that the decreased rate of synthesis of ODC was attributable to almost unaltered amounts of mRNA in drug-treated cells, thus supporting the view that especially GBG apparently depressed the expression of ODC at some post-transcriptional level.
The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+
Authors: Farese R V , Davis J S , Barnes D E , Standaert M L , Babischkin J S , Hock R , Rosic N K , Pollet R J
Published: August 2015

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated ‘second messenger’ which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular ‘messenger’ for controlling metabolic processes during insulin action.
Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization
Authors: Opdenakker G , Billiau A , Volckaert G , de Somer P
Published: August 2015

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.