bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity
Authors: Evans S W , Rennick D , Farrar W L
Published: August 2015

The haematopoietic growth factors multi-colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2 specifically control the production and proliferation of distinct leucocyte series. Each growth factor acts on a unique surface receptor associated with an appropriate signal-transduction apparatus. In this report we identify a 68 kDa substrate which is phosphorylated after stimulation of different cell types with multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2. The 68 kDa substrate is also phosphorylated in each cell line stimulated with synthetic diacylglycerol, a direct activator of protein kinase C. Interestingly, granulocyte/macrophage colony-stimulating factor does not induce phosphorylation of the 68 kDa molecule. The 68 kDa molecule that is phosphorylated after stimulation with each ligand yielded similar peptide maps after chymotryptic digestion; furthermore, the substrate was always phosphorylated on threonine residues. Phosphorylation of the same residues in the 68 kDa substrate suggests that activation of protein kinase C is one common signal-transduction event associated with the action of multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2.
An abundant and novel protein of 22 kDa (SM22) is widely distributed in smooth muscles. Purification from bovine aorta
Authors: Lees-Miller J P , Heeley D H , Smillie L B
Published: August 2015

Using a rabbit polyclonal-antibody preparation directed against the chicken gizzard protein, we demonstrated by immunoblotting the presence of the 22 kDa protein (SM22) in a variety of chicken smooth-muscle-containing organs, including uterus, intestine, gizzard, oesophagus and aorta. Protein SM22 was present in only trace amounts in brain, liver and heart, and could not be detected in chicken breast muscle. The antibody preparation did not cross-react with extracts of bovine aorta. However, the presence of SM22 as a major component in bovine aorta and pig carotid was demonstrated by its co-migration with the purified chicken gizzard protein on one- and two-dimensional polyacrylamide electrophoretic gels. Its molar abundance relative to actin was estimated to be 0.9:6.0 and 1.4:6.0 for bovine aorta and pig carotid respectively. Like the chicken gizzard protein, it separates on pH-gradient electrophoresis into at least three variants, alpha, beta and gamma, with similar apparent Mr. Purification of the aorta SM22 showed it to have a similar amino acid composition to the chicken gizzard protein. We conclude that SM22 is widely distributed and an abundant and unique protein component of smooth-muscle tissues of birds and mammals.
Intermediate forms of human β-N-acetylhexosaminidase lack activity towards 4-methylumbelliferyl β-N-acetylglucosaminide 6-sulphate
Authors: Beccari T , Emiliani C , Hosseini R , Orlacchio A , Stirling J L
Published: August 2015

4-Methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate was purified from a mixture containing its unsulphated precursor. The substrate was used to test for the presence of functional alpha-subunits in ‘intermediate’ forms of human beta-N-acetylhexosaminidase in samples of normal and pregnancy serum and in extracts of placenta and lymphocytes from a patient with common acute lymphoblastic leukaemia. Intermediate forms in these samples had no activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate, indicating that they lack alpha-subunits.
An isotopic method for measurement of muscle protein synthesis and degradation in vivo
Authors: Barrett E J , Revkin J H , Young L H , Zaret B L , Jacob R , Gelfand R A
Published: August 2015

In eight anaesthetized post-absorptive dogs we measured the concentration and specific radioactivity of phenylalanine and leucine in arterial and femoral-venous plasma, together with hindlimb flow during a continuous infusion of L-[ring-2,6-3H]phenylalanine and [1-14C]leucine. The femoral-venous plasma concentration was greater than arterial for both phenylalanine and leucine (P less than 0.05 for each). Despite net amino acid release there was a significant removal of both labelled phenylalanine and labelled leucine. Consequently, a significant dilution of specific radioactivity was observed between artery and vein for both radio-tracers. The uptake of leucine from the arterial circulation by the hindlimb exceeded by 2.6-fold that of phenylalanine; the measured molar ratio of leucine to phenylalanine in hindlimb muscle protein averaged 2.4 +/- 0.1. Since phenylalanine is neither synthesized nor degraded by muscle tissue, the measured removal of tracer and the dilution of tracer specific radioactivity across the hindlimb can be used to estimate rates of phenylalanine incorporation into, and release from, tissue protein. The estimated rate of protein synthesis by hindlimb averaged 644 +/- 250 nmol of phenylalanine/min. This was exceeded by the rate of tissue protein degradation (987 +/- 285 nmol of phenylalanine/min). The present results demonstrate that the dilution of the specific radioactivity of labelled phenylalanine can be readily measured across dog hindlimb. This measurement, coupled with an estimate of tissue blood flow, can provide a readily measured, non-destructive, method for estimation of protein turnover in specific muscle beds in vivo. Measurements can be made repeatedly over time in a single experiment, allowing the study of factors which regulate protein turnover. The method developed here in dogs can be readily extended to clinical studies.
Distinction between ‘A’-esterases and arylesterases. Implications for esterase classification
Authors: Mackness M I , Thompson H M , Hardy A R , Walker C H
Published: August 2015

‘A’-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable ‘A’-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both ‘A’- and ‘aryl’-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not ‘A’-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications.
The inhibition of infectivity of bacteriophage ϕ X174 by high-valency metal cations and cyclic polyamines
Authors: Rowatt E , Williams R J P
Published: August 2015

It has been shown previously that many aliphatic polyamines and metal cations decrease the infectivity of bacteriophage phi X174 when lipopolysaccharide from Escherichia coli C is present. Cations of higher charge are more effective. In the present paper it is shown that certain of the metal cations and cyclic polyamines diminish phage infectivity without lipopolysaccharide. The relation of cation concentration to loss of infectivity is different for the two types of reaction. In the absence of lipopolysaccharide the inhibition increases with the charge of the cation, but that by cyclic polyamines depends also on the hydrocarbon chains and their conformation. Some characteristics of the reactions are discussed.
Glucose metabolism in isolated brown adipocytes under β-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis
Authors: Isler D , Hill H P , Meier M K
Published: August 2015

To quantify the potential of brown adipose tissue as a target organ for glucose oxidation, O2 consumption and glucose metabolism in isolated rat brown adipocytes were measured in the presence and absence of insulin, by using the beta-agonists isoprenaline or Ro 16-8714 to stimulate thermogenesis. Basal metabolic rate (278 mumol of O2/h per g of lipid) was maximally stimulated with isoprenaline (20 nm) and Ro 16-8714 (20 microM) to 1633 and 1024 mumol of O2/h per g respectively, whereas insulin had no effect on O2 consumption. Total glucose uptake, derived from the sum of [U-14C]glucose incorporation into CO2 and total lipids and lactate release, was enhanced with insulin. Isoprenaline and Ro 16-8714 had no effect on insulin-induced glucose uptake, but promoted glucose oxidation while inhibiting insulin-dependent lipogenesis and lactate production. A maximal value for glucose oxidation was obtained under the combined action of Ro 16-8714 and insulin, which corresponded to an equivalent of 165 mumol of O2/h per g of lipid. This makes it clear that glucose is a minor substrate for isolated brown adipocytes, fuelling thermogenesis by a maximum of 16%.
Metabolism of d-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate
Authors: Shears S B , Parry J B , Tang E K , Irvine R F , Michell R H , Kirk C J
Published: August 2015

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5′-[beta gamma-imido]triphosphate, adenosine 5′-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5′-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.
Non-specific peptide size effects in the recognition by site-specific T-cell clones. Demonstration with a T site of myoglobin
Authors: Atassi M Z , Yoshioka M , Bean M , Bixler G S
Published: August 2015

Six regions (T sites) of myoglobin (Mb) were found by a comprehensive synthetic strategy to stimulate Mb-primed lymph-node cells. To define precisely the N-terminal boundary of the immunodominant T site (residues 107-120) with site-specific T-cell clones and to determine the effects of peptide size on their stimulation, two sets of peptides were employed. In one set, the peptides were elongated to the left from His-113 by one-residue increments of the Mb sequence. The other set represented an identical stepwise elongation by one-residue increments of the Mb sequence, but which were extended by additional unrelated (‘nonsense’) residues to a uniform size of 14 residues. Examination of the proliferative responses of eight T-cell clones, derived from Mb-primed DBA/2 (H-2d) or SJL (H-2s) mice, revealed a dramatic non-specific size requirement. In every clone, the longer nonsense-extended peptides achieved maximum stimulating activity at a lower optimum peptide dose than its natural-sequence, but shorter, analogue. In addition, slight (one-residue) differences in the N-terminal boundaries among the clones was observed. Thus, the fine specificity of each clone was mapped to the region from residue 111 or 112 to about residue 120 of Mb, which coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.
Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium
Authors: Gustin M C , Nelson D L
Published: August 2015

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.
The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains
Authors: Duncan K , Edwards R M , Coggins J R
Published: August 2015

The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes.
Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre
Authors: Ashby G A , Thorneley R N F
Published: August 2015

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 × 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 × 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 × 10(5) less than k less than 3 × 10(6) M-1.s-1 and protein conformation change 6 × 10(2) less than k less than 6 × 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.
Preventive effect of malotilate on carbon tetrachloride-induced liver damage and collagen accumulation in the rat
Authors: Ala-Kokko L , Stenbäck F , Ryhänen L
Published: August 2015

Malotilate is a new drug suggested for use in chronic liver diseases. It is shown here to prevent liver damage caused by CCl4. The concomitant administration of malotilate with CCl4 significantly decreased hydroxyproline accumulation in the liver, liver prolyl 4-hydroxylase and liver and serum galactosylhydroxylysyl glucosyltransferase activities. However, it had no effect on the daily urinary hydroxyproline excretion or the hydroxyproline content of the skin, liver or lungs in normal young growing rats. It also had no specific inhibitory effect on hydroxyproline synthesis or secretion in fibroblast cultures, and did not affect the amount of procollagen-alpha 1(I)-specific mRNAs in these cultures. Thus it seems to have no direct inhibitory effect on collagen metabolism. In addition to inhibition of liver collagen accumulation, malotilate was also able to prevent the development of morphological changes in the liver such as focal necrosis, fatty infiltration and inflammatory changes. It also normalized almost completely the standard liver-function tests. It is possible that malotilate may prevent excessive collagen deposition by inhibiting the inflammation caused by CCl4-induced liver damage.
Identification and characterization of the glucose-transport protein of the bovine blood/brain barrier
Authors: Kasanicki M A , Cairns M T , Davies A , Gardiner R M , Baldwin S A
Published: August 2015

The glucose-transport protein from bovine cerebral-cortex microvessels has been identified and characterized by virtue of its ability to bind the ligand [4-3H]cytochalasin B. Microvessel membranes were found to contain a single set of glucose-inhibitable high-affinity cytochalasin B-binding sites [113 +/- 16 (S.E.M.) pmol/mg of membrane protein], with an association constant of 6.8 +/- 1.8 (S.E.M.) micron-1. D-Glucose inhibited the binding to these sites with a Ki of 31 mM. The transport protein was identified by photoaffinity labelling with [4-3H]cytochalasin B and was found to migrate as a broad band of apparent Mr 55,000 on SDS/polyacrylamide gels. Labelling was inhibited by D-glucose, but not by L-glucose. Treatment with endoglycosidase F yielded a sharper band of apparent Mr 46,000, indicating that the transport protein is glycosylated. However, in contrast with the human erythrocyte glucose transporter, digestion with endo-beta-galactosidase had little effect on the electrophoretic mobility of the microvessel protein. Tryptic digestion of the photolabelled protein yielded a radioactive fragment of apparent Mr 18,000, similar to that of the fragment produced by digestion of the labelled human erythrocyte glucose transporter. In addition, a protein of Mr identical with that of the photolabelled transporter was labelled on Western blots of microvessel membranes by antisera raised against the intact erythrocyte transporter and against synthetic peptides corresponding to its N- and C-terminal regions. It is concluded that the glucose-transport protein of bovine cerebral-cortex microvessel endothelial cells shows structural homology with the human erythrocyte glucose transporter.
An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88° C
Authors: Cowan D A , Smolenski K A , Daniel R M , Morgan H W
Published: August 2015

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.
A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine
Authors: Said H M , Redha R
Published: August 2015

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.
Epidermal growth factor, like glucagon, exerts a short-term stimulation of alanine transport in rat hepatocytes
Authors: Moule S K , McGivan J D
Published: August 2015

Epidermal growth factor causes a transient stimulation of alanine transport in hepatocytes. The stimulation is maximal after 30 min, and the rate returns to the control value after 90 min. These characteristics are very similar to the short-term stimulation of alanine transport by glucagon, which has been attributed to cell membrane hyperpolarization.
Light-induced changes in the content of inositol phosphates in squid (Loligo pealei) retina
Authors: Brown J E , Watkins D C , Malbon C C
Published: August 2015

Illumination induced an increase in inositol polyphosphates, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, in the photoreceptors of the squid, Loligo pealei. There was a concomitant decrease in phosphatidylinositol 4,5-bisphosphate, but no light-induced change in any other phosphoinositide. None of these stimulus-induced changes were altered by treatment in vivo with pertussis toxin, which ADP-ribosylated a Mr-39000-peptide. These findings support the hypothesis that inositol 1,4,5-trisphosphate participates as either a messenger or a modulator in transduction in invertebrate photoreceptors.
Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures
Authors: Chowdhury M H , Agius L
Published: August 2015

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.
Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src
Authors: Powell M A , Glenney J R
Published: August 2015

Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.
Uptake and efflux of copper-64 in Menkes'-disease and normal continuous lymphoid cell lines
Authors: Herd S M , Camakaris J , Christofferson R , Wookey P , Danks D M
Published: August 2015

The accumulation of copper over 2 h by normal lymphoid cells and those from Menkes'-disease patients (Menkes' cells) was found to be biphasic, with an initial phase of rapid uptake, an approach to steady state at around 40-60 min, followed by a further accumulation phase. The accumulation of copper was not diminished by the addition of a variety of metabolic inhibitors, suggesting that copper uptake is not an active process. The presence of carbonyl cyanide m-chlorophenylhydrazone in the culture medium stimulated the uptake and accumulation of copper in both normal and Menkes' cells to the same absolute level. This effect appeared to be specific for copper, since the accumulation of Zn and Cd was unaffected. Menkes' cells did not differ from normal in their initial rate of copper uptake. Analysis of the uptake curve suggested that the membrane transport of copper involves both passive and facilitated diffusion. Initial rate of efflux from the cells was approximated by two methods. Menkes' cells did not appear to be affected in this function. It seems likely that the basic defect in Menkes' disease involves a step in intracellular copper transport rather than the membrane transport of copper.
Differential permeabilization of membranes by saponin treatment of isolated rat hepatocytes. Release of secretory proteins
Authors: Wassler M , Jonasson I , Persson R , Fries E
Published: August 2015

Monolayer cultures of rat hepatocytes were treated with increasing concentrations of saponin (prepared from Gypsophila plants) for 30 min at 6 degrees C. Differential permeabilization of the intracellular membranes could be demonstrated: at 0.040 mg of saponin/ml the plasma membrane was permeabilized, as assessed by the release of 50% of the total cellular amount of lactate dehydrogenase, and at 0.20 mg/ml the endoplasmic reticulum was permeabilized, as measured by the release of 50% of pulse-35S-labelled albumin. The Golgi complex was permeabilized at an intermediate saponin concentration, as indicated by the release of homogeneously 35S-labelled albumin; about half the intracellular albumin is located in this organelle. At 1.0 up to 5.0 mg of saponin/ml 90-95% of the radioactively labelled albumin was released. Even at 5.0 mg/ml less than 10% of the membrane of the endoplasmic reticulum was solubilized, as judged by the degree of release of a membrane-bound enzyme specific for this organelle. These results demonstrate the usefulness of saponin as a tool for investigating the interior of different intracellular compartments.
Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis
Authors: Watanabe F , Nakano Y , Kitaoka S
Published: August 2015

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.
Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture
Authors: Castillo C J , Colburn P , Buonassisi V
Published: August 2015

We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.
Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding
Authors: Morris G E , Frost L C , Newport P A , Hudson N
Published: August 2015

(1) The binding sites of two monoclonal antibodies, CK-2A7 and CK-5H5, have been located to a 60-amino-acid sequence in the N-terminal region of creatine kinase (CK) by the use of chemical cleavage with formic acid (which cleaves proteins at Asp-Pro bonds) and cyanogen bromide (which cleaves at Met residues). (2) A simple method for preparing chemically-cleaved fragments of proteins for electrophoresis and Western blotting is described. (3) Binding studies with CK preparations from different animal species show that single amino acid changes at residues 39 or 82 prevent binding of CK-2A7 and CK-5H5 respectively. We suggest that Lys-39 and Glu-82 form parts of the binding sites on CK for the two monoclonal antibodies. The two sites lie in variable regions at each end of a highly-conserved sequence (residues 46 to 79) and are inaccessible to antibody in the native enzyme. (4) One of the antibodies, CK-2A7, inhibits the refolding of CK to native enzyme after denaturation by urea.
Hepatic uptake of amino acids in late-pregnant rats. Effect of food deprivation
Authors: Casado J , Remesar X , Pastor-Anglada M
Published: August 2015

Hepatic availability, uptake and fractional extraction of amino acids were estimated in anaesthetized 21-day-pregnant and age-matched virgin rats, either fed or after 24 h starvation. Amino acid availability was unaltered in fed pregnant rats as compared with fed virgin controls. However, the hepatic uptake of these compounds was higher in the former than in the latter. These adaptations were mediated by an increase in the hepatic capability to take up amino acids in late-pregnant rats, as reflected by the changes found for the fractional extraction rates. The decrease in amino acid availability found after starvation was more pronounced in pregnant than in virgin rats. Nevertheless, the hepatic uptake was similar in both groups. These results indicate that amino acids are not limiting for ureagenesis during late pregnancy, strongly suggesting that the mechanism(s) which modulate urea synthesis may be intracellular in origin.
Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein
Authors: Wu J J , Eyre D R , Slayter H S
Published: August 2015

The collagen framework of the intervertebral disc contains two major fibril-forming collagens, types I and II. Smaller amounts of other types of collagen are also present. On examination of the nature and distribution of these minor collagens within bovine disc tissue, type VI collagen was found to be unusually abundant. It accounted for about 20% of the total collagen in calf nucleus pulposus, and about 5% in the annulus fibrosus. It was discovered by serially digesting disc tissue with chondroitin ABC lyase and Streptomyces hyaluronidase that native covalent polymers of type VI collagen could be extracted. Electron micrographs of this material prepared by rotary shadowing revealed the characteristic dimensions of tetramers and double tetramers of type VI molecules, with their central rods and terminal globular domains. Molecular-sieve column chromatography on agarose under non-reducing non-denaturing conditions gave a series of protein peaks with molecular sizes equivalent to the tetramer, double tetramer and higher multimers. On SDS/polyacrylamide-gel electrophoresis after disulphide cleavage, these fractions of type VI collagen all showed a main band at Mr 140,000 and four lesser bands between Mr 180,000 and 240,000. On electrophoresis without disulphide cleavage in agarose/2.4% polyacrylamide only dimeric (six chains) and tetrameric (12 chains) forms of type VI molecules were present. The ability to extract all the type VI collagen of the tissue in 4 M-guanidinium chloride, and absence of aldehyde-mediated cross-linking residues on direct analysis, showed that, in contrast with most matrix collagens, type VI collagen does not function as a covalently cross-linked structural polymer.
Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes
Authors: Davis F B , Davis P J , Blas S D , Schoenl M
Published: August 2015

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.
Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2
Authors: Lukács G L , Kapus A
Published: August 2015

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.
Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol
Authors: Kramer R M , Checani G C , Deykin D
Published: August 2015

We examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated 2.5 microM-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A2 activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to 29 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonoylglycerol. 1-Stearoyl-2-arachidonoylglycerol at concentrations of 2-5 mol% increased phospholipase A2 activity 1.3-1.8-fold. Exogenously added 1-oleoyl-2-acetylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A2. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca2+-activated phospholipase A2.
Profile of the α-bungarotoxin-binding regions on the extracellular part of the α-chain of Torpedo californica acetylcholine receptor
Authors: MULAC-JERIČEVIČ B , Atassi M Z
Published: August 2015

The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different ‘faces’ in one (or more) sites.
A study of the interaction between cartilage proteoglycan and link protein
Authors: Thornton D J , Sheehan J K , Nieduszynski I A
Published: August 2015

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two ‘dissociative’ density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.
Occurrence of unusual molecular species of sphingomyelin containing 28-34-carbon polyenoic fatty acids in ram spermatozoa
Authors: Poulos A , Johnson D W , Beckman K , White I G , Easton C
Published: August 2015

The high levels of very long chain fatty acids found in ram spermatozoa are located almost exclusively in one of two separable species of sphingomyelin. Mass spectral analysis, including fast atom bombardment of the purified sphingomyelin, has shown the fatty acids to have a carbon chain length of between 28 and 34, with between four and six double bonds, and to belong predominantly to the n-3 series.
The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles. Measurement of vesicle fusion by fluorescence energy transfer
Authors: Comerford J G , Dawson A P
Published: August 2015

1. GTP-promoted fusion between microsomal vesicles was studied by using fluorescence-resonance-energy transfer between the fluorescent membrane probes octadecanoyl-aminofluorescein and octadecyl-rhodamine. 2. The fluorescence increase after GTP addition does not require the presence of ATP, is unaffected by changes in free [Ca2+] in the range 10 microM-1 nM, but requires Mg2+, although higher Mg2+ concentrations are inhibitory. 3. In terms of requirements for poly(ethylene glycol), dependence on GTP concentration and inhibition by high Mg2+ concentrations, there is excellent correlation between rate of increase in fluorescence and rate of GTP-promoted Ca2+ efflux measured under Ca2+ transport conditions. 4. The observations support our previous conclusions that GTP-induced membrane fusion plays a major role in causing GTP-promoted Ca2+ efflux from microsomal vesicles.