bluuebunny/crossref_metadata_2025 huggingface dataset:
An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity
Authors: Edwards R J , Singleton A M , Murray B P , Sesardic D , Rich K J , Davies D S , Boobis A R
Published: August 2015

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.
Diabetes abolishes the GTP-dependent, but not the receptor-dependent inhibitory function of the inhibitory guanine-nucleotide-binding regulatory protein (Gi) on adipocyte adenylate cyclase activity
Authors: Strassheim D , Milligan G , Houslay M D
Published: August 2015

Adipocyte membranes from control rats exhibited a functional Gi (inhibitory guanine-nucleotide-binding protein) activity which could be assessed either by the inhibitory action of low concentrations of guanosine 5-[beta gamma-imido]triphosphate (p[NH]ppG) upon forskolin-stimulated adenylate cyclase activity or by the inhibitory action of high concentrations of GTP upon isoprenaline-stimulated adenylate cyclase activity. When membranes from animals made diabetic with streptozotocin were used, then both such inhibitory functions of Gi were abolished. In contrast, receptor-mediated inhibitory responses of Gi, effected by N6-phenylisopropyl (adenosine), prostaglandin E2 or nicotinate, were either unchanged or even apparently more effective in membranes from diabetic animals. Induction of diabetes did not cause any change in the adipocyte plasma membrane levels of the alpha, GTP-binding subunits of either Gi1 or Gi2 or of Gs (stimulatory guanine-nucleotide-binding protein), but elicited an increase in the level of alpha-Gi3. The induction of diabetes reduced the specific activity of adenylate cyclase in adipocyte membranes and enhanced the stimulatory effect of isoprenaline. It is suggested that diabetes causes selective changes in the functioning of Gi in adipocyte membranes which removes the tonic GTP-dependent inhibitory function of this G-protein.
A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition
Authors: Templeton W , Kowlessur D , Thomas E W , Topham C M , Brocklehurst K
Published: August 2015

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)
Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells
Authors: Conner J , Vallet-Collom I , Daveau M , Delers F , Hiron M , Lebreton J P , Guillouzo A
Published: August 2015

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.
Cobalamin neuropathy. Is S-adenosylhomocysteine toxicity a factor?
Authors: Vieira-Makings E , Metz J , Van der Westhuyzen J , Bottiglieri T , Chanarin I
Published: August 2015

Cobalamin neuropathy was produced in cape fruit bats (Rousettus aegyptiacus) by a cobalamin-free diet combined with intermittent exposure to nitrous oxide, which inactivates cobalamin. There were no significant differences in S-adenosylmethionine/S-adenosylhomocysteine ratios in the central nervous system of cobalamin-deficient and cobalamin-replete bats. Taken with other data there are no grounds of support for a hypothesis that cobalamin neuropathy is the result of impaired methylation, however produced.
Regulation of total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscle incubated flaccid or at resting length
Authors: Hasselgren P O , Hall-Angerås M , Angerås U , Benson D , James J H , Fischer J E
Published: August 2015

The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.
Oral administration of vanadate to streptozotocin-diabetic rats restores the glucose-induced activation of liver glycogen synthase
Authors: Bollen M , Miralpeix M , Ventura F , Toth B , Bartrons R , Stalmans W
Published: August 2015

Isolated hepatocytes from streptozotocin-diabetic rats failed to respond to a glucose load with an activation of glycogen synthase. This lesion was associated with severely decreased activities of glycogen-synthase phosphatase and of glucokinase. All these defects were abolished after consumption for 13-18 days of drinking water containing Na3VO4 (0.7 mg/ml), and they were partially restored after 3.5 days, when the blood glucose concentration was already normalized. In all conditions the maximal extent of activation of glycogen synthase in cells closely parallelled the activity of glycogen-synthase phosphatase.
Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis
Authors: Jones J , Hallett M B , Morgan B P
Published: August 2015

The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.
The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue
Authors: Armstrong M K , Bernlohr D A , Storch J , Clarke S D
Published: August 2015

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.
Effects of ionizing radiations on proteins. Evidence of non-random fragmentations and a caution in the use of the method for determination of molecular mass
Authors: Le Maire M , Thauvette L , de Foresta B , Viel A , Beauregard G , Potier M
Published: August 2015

We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.
Non-proteolytic activation of cellular protransglutaminase (placenta macrophage factor XIII)
Authors: Polgár J , Hidasi V , Muszbek L
Published: August 2015

Plasma Factor XIII is a zymogen (plasma protransglutaminase) with the tetrametric structure A2B2, whereas the cellular protransglutaminase, i.e. Factor XIII in the platelet and monocyte/macrophage, consists exclusively of A subunits (A2). It is generally accepted that at Ca2+ concentrations comparable with that in plasma the proteolytic removal of an N-terminal activation peptide is the prerequisite for the Ca2(+)-induced formation of a catalytically active configuration of subunit A. In this study it was demonstrated that at high concentrations NaCl or KCl induced a non-proteolytic activation of cellular (placental macrophage) but not plasma protransglutaminase. The activation depended on time and salt concentration, and Ca2+, in the range 0-20 mM, greatly enhanced the activation process. At 1.25 M-NaCl maximal activation occurred within 60 min in the presence of 2 mM-CaCl2, and even at physiological NaCl concentration a slow progressive activation could be observed in the presence of Ca2+. The specific activity of salt-activated Factor XIII was 1.5-2.0-fold higher than that obtained after thrombin activation. The non-proteolytic activation of cellular protransglutaminase was abolished by the addition of subunit B of plasma Factor XIII in stoichiometric amount, which suggests that (one of) the physiological function(s) of the B subunit in plasma Factor XIII is to prevent the slow spontaneous activation of A subunit that would occur in a plasmatic environment.
Myelin basic protein is affected by reduced synthesis of myelin proteolipid protein in the jimpy mouse
Authors: Fannon A M , Moscarello M A
Published: August 2015

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.
Spatially resolved changes in diabetic rat skeletal muscle metabolism in vivo studied by 31P-n.m.r. spectroscopy
Authors: Challiss R A J , Blackledge M J , Radda G K
Published: August 2015

Phase-modulated rotating-frame imaging (p.m.r.f.i.), a localization technique for 31P-n.m.r. spectroscopy, has been applied to obtain information on the heterogeneity of phosphorus-containing metabolites and pH in the skeletal muscle of control and streptozotocin-diabetic rats. Using this method, the metabolic changes in four spatially resolved longitudinal slices (where slice I is superficial and slice IV is deep muscle) through the ankle flexor muscles have been investigated at rest and during steady-state isometric twitch-contraction at 2 Hz. At rest, intracellular pH was lower, and phosphocreatine (PCr)/ATP was higher, throughout the muscle mass in diabetic compared with control animals. The change in PCr/ATP in diabetic muscle correlated with a decrease in the chemically determined ATP concentration. During the muscle stimulation period, the decrease in pH observed in diabetic muscle at rest was maintained, but not exacerbated, by the contractile stimulus. Stimulation of muscle contraction caused more marked changes in PCr/(PCr + Pi), PCr/ATP and Pi/ATP in the diabetic group. These changes were most evident in slice III, which contains the greatest proportion of fast glycolytic-oxidative (type IIa) fibres, in which statistically significant differences were observed for all metabolite ratios. The results presented suggest that some degree of heterogeneity occurs in diabetic skeletal muscle in vivo with respect to the extent of metabolic dysfunction caused by the diabetic insult and that regions of the muscle containing high proportions of type IIa fibres appear to be most severely affected.
Comparative study of the glycosylation of platelet glycoprotein GPIIb/IIIa and the vitronectin receptor. Differential processing of their β-subunit
Authors: Troesch A , Duperray A , Polack B , Marguerie G
Published: August 2015

The platelet glycoprotein GPIIb/IIIa and the vitronectin receptor (VNR) are alpha beta-heterodimeric proteins and share the same beta-subunit. By performing swainsonine treatment and digestion with endoglycosidase H (Endo H), we showed that the heavy chains of GPIIb and VNR alpha are glycosylated by complex-type oligosaccharide chains, and provided the first evidence for the presence of one complex carbohydrate residue on their light chains. The proteolytic cleavage of pro-GPIIb and the acquisition of Endo H-resistance are independent events occurring in the same Golgi compartment. We demonstrated the Endo H-sensitivity of GPIIIa and VNR beta in all cellular systems tested. In addition, this beta-subunit is differently glycosylated according to whether it is associated with GPIIb or VNR alpha, one carbohydrate chain being processed to the complex type on GPIIIa, but not on VNR beta.
Modulation of phospholipase A2 activity by the tumour promoters phorbol esters and teleocidin
Authors: Nam K Y , Morino A , Kimura S , Fujiki H , Imanishi Y
Published: August 2015

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.
Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines
Authors: Krueger M J , McKeown K , Ramsay R R , Youngster S K , Singer T P
Published: August 2015

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2′-ethyl- and 3′-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.
4-O-(1-carboxyethyl)-l-rhamnose, a second unique acidic sugar found in an extracellular polysaccharide from Butyrivibrio fibrisolvens strain 49
Authors: Stack R J , Weisleder D
Published: August 2015

Butyrivibrio fibrisolvens strain 49 excretes a polysaccharide that contains D-glucose, D-galactose, 4-O-(1-carboxyethyl)-D-galactose, and an acidic component of previously unknown structure. We report here the identity of the unknown as 4-O-(1-carboxyethyl)-L-rhamnose. The structure of this previously unknown compound was deduced from (1) comprehensive electron-impact and chemical-ionization mass-spectroscopic studies of differentially labelled derivatives prepared from the unknown, (2) 13C-n.m.r. and 1H-n.m.r. studies of purified neutral sugars derived from the unknown and (3) chemical degradation experiments.
Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor
Authors: Ely J A , Hunyady L , Baukal A J , Catt K J
Published: August 2015

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.
Decarboxylation of uroporphyrinogen III by erythrocyte uroporphyrinogen decarboxylase. Evidence for a random decarboxylation mechanism
Authors: Luo J , Lim C K
Published: August 2015

The isomeric composition of type-III heptacarboxylic porphyrinogens derived from decarbosylation of uroporphyrinogen III by erythrocyte uroporphyringogen decarboxylase was analysed by h.p.l.c. with electrochemical detection. All four possible isomers were identified, and there were little differences in the proportion of isomers formed by erythrocytes from normal subjects and from patients with sporadic porphyria cutanea tarda. The results provide conclusive evidence that the normal decarboxylation pathway is random in nature, and the fourth isomer only increases when enzyme abnormality is found.
A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins
Authors: Jefferis R , Lund J , Mizutani H , Nakagawa H , Kawazoe Y , Arata Y , Takahashi N
Published: August 2015

Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.
Thyroid status and β-agonistic effects on cytosolic calcium concentrations in single rat cardiac myocytes activated by electrical stimulation or high-K+ depolarization
Authors: Beekman R E , van Hardeveld C , Simonides W S
Published: August 2015

The effects of the thyroid status on the cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were studied at rest and during contraction. The mean resting [Ca2+]i increased significantly from the hypothyroid (45 +/- 4 nM) through the euthyroid (69 +/- 12 nM) to the hyperthyroid condition (80 +/- 11 nM) at extracellular Ca2+ concentrations ([Ca2+]o) up to 2.5 mM. At [Ca2+]o above 2.5 mM the differences in [Ca2+]i between the groups became less. The amplitude of the Ca2+ transients became higher in all groups with increasing [Ca2+]o (1, 2.5 and 5 mM), and was highest at all [Ca2+]o in hyperthyroid myocytes. The beta-agonist isoprenaline elevated peak [Ca2+]i during contraction and increased the rate of the decay of the Ca2+ transients to a greater extent in hypothyroid myocytes than in hyperthyroid myocytes. Depolarization with high [K+]o induced a large but transient [Ca2+]i overshoot in hypothyroid myocytes, but not in hyperthyroid myocytes, before a new elevated steady-state [Ca2+]i was reached, which was not different between the groups. When isoprenaline was added to K+ o-depolarized myocytes after a steady state was reached, a significantly larger extra increase in [Ca2+]i was measured in the hypothyroid group (28%) compared with the hyperthyroid group (8%). It is concluded that in cardiac tissue exposed to increasing amounts of thyroid hormones (1) [Ca2+]i increases at rest and during contraction in cardiomyocytes and (2) interventions which favour Ca2+ entry into the cytosol [( Ca2+]o elevation, high [K+]o, beta-agonists) tend to have less impact on Ca2+ homoeostasis.
Post-translational processing of chicken bone phosphoproteins. Identification of bone (phospho)protein kinase
Authors: Mikuni-Takagaki Y , Glimcher M J
Published: August 2015

We have detected a protein kinase which phosphorylates bone phosphoproteins (BPPs) in the detergent extract of the membranous fractions in the periosteal bone strips of 12-day-embryonic-chick tibia. This enzyme, tentatively named BPP kinase, has a catalytic subunit of Mr approximately 39,000, utilizes GTP as well as ATP as a phospho-group donor, is inhibited by 2,3-bisphosphoglycerate and heparin, and is therefore similar to casein kinase II. The enzyme can phosphorylate dephosphorylated proteins such as casein, phosvitin and chicken BPPs, but the last-named are preferred substrates. The in vitro-phosphorylation-assay products of this enzyme in the extract were indistinguishable on an SDS/polyacrylamide gel from the major [32P]phosphoproteins metabolically labelled in the embryonic-chick bone tissue. The regulatory mechanisms of the phosphorylation process of BPPs by BPP kinase as well as the potential role of this enzyme in mineralization are discussed.
Reciprocal changes in amino acid metabolism in mammary gland and liver of the lactating rat on starvation and refeeding as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate
Authors: Tedstone A E , Ilic V , Williamson D H
Published: August 2015

Measurements of the tissue accumulation in vivo and in vitro by hepatocytes and mammary-gland acini of alpha-amino[1-14C]isobutyrate ([1-14C]AIB) were compared in virgin and lactating rats. The results indicate the existence of a reciprocal relationship between mammary gland and liver for AIB accumulation that is dependent on the lactational and the nutritional state of the rat. This suggests that amino acids are preferentially directed to the mammary gland during active lactation.
Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize γ-aminobutyraldehyde to γ-aminobutyric acid
Authors: Abe T , Takada K , Ohkawa K , Matsuda M
Published: August 2015

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5′-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.
Urokinase- and tissue-type plasminogen activators are suppressed by cortisol in the involuting prostate of castrated rats
Authors: Freeman S N , Rennie P S , Chao J , Lund L R , Andreasen P A
Published: August 2015

The effects of cortisol on the inhibition of cell-death processes and suppression of plasminogen-activator (PA) activity during involution of the rat ventral prostate gland were investigated to determine the principal type of PA activated by castration and inhibited by this hormone and whether the mechanism responsible for decreased PA activity involved reductions in enzyme synthesis or increased activity of a PA inhibitor. By using the technique of fibrin-agarose zymography, three bands of PA activity were detected at 4 and 7 days after castration: a major band with a molecular mass of approx. 30 kDa and two minor bands of 48 kDa and 64 kDa. Both the 30 kDa and 48 kDa activities were inhibited with anti-[urokinase-type PA (u-PA)] IgG. The 64 kDa activity was inhibited by anti-[tissue-type PA (t-PA)] IgG. In addition to retarding prostatic involution, daily administration of cortisol to the castrated animals suppressed all three bands of PA activity. A comparison of the pattern of total PA activity and of e.l.i.s.a. estimates of u-PA concentration during the castration-induced rise and after cortisol inhibition indicated a near perfect correlation between the two parameters. Northern-blot analysis using prostatic polyadenylated RNA revealed that the level of u-PA mRNA was highest at 4 and 7 days after castration and that cortisol treatment repressed u-PA mRNA to a level similar to that in non-castrated controls. Neither Northern hybridizations nor reverse zymography detected RNA transcripts or activity corresponding to the PA inhibitor PAI-1 in any of the prostate samples. Western-blot analysis revealed that, although the amount of arginine esterase A, another prostatic proteinase, also increased after castration, the rise in concentration of this protein was not blocked by glucocorticoid administration. Together our findings indicate the following: (1) the predominant form of PA activity induced in the prostate after castration and inhibited by cortisol is a 30 kDa form of u-PA. Although less prominent, t-PA and a 48 kDa form of u-PA follow a similar pattern of induction and inhibition; (2) changes in u-PA activity in response to castration and cortisol treatment are due to alterations in the level of u-PA mRNA and protein rather than in the activity of PAI-1; (3) not all castration-induced proteinases in the prostate are inhibited by cortisol.
Metabolic effects of platelet-activating factor in rats in vivo. Stimulation of hepatic glycogenolysis and lipogenesis
Authors: Evans R D , Ilic V , Williamson D H
Published: August 2015

1. The effects of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine; PAF) on hepatic metabolism in vivo in rats were studied. 2. PAF stimulated synthesis of hepatic lipid (saponified and non-saponified) in a dose-dependent fashion and caused hypertriglyceridaemia. There was no effect of PAF on lipogenesis in isolated hepatocytes. 3. High doses of PAF also decreased hepatic glycogen. 4. All doses of PAF decreased plasma insulin, and this was accompanied by hyperglycaemia, except at the lowest dose. 5. The selective PAF-receptor antagonist L659.989 prevented the stimulation of lipogenesis, but indomethacin did not.
Conditions that result in the mobilization and influx of Ca2+ into rat hepatocytes induce the rapid loss of 3-hydroxy-3-methylglutaryl-CoA reductase activity that is not reversed by phosphatase treatment
Authors: Zammit V A , Caldwell A M
Published: August 2015

We investigated the effects of conditions that induce Ca2+ mobilization from intracellular stores and Ca2+ influx into hepatocytes on the expressed and total (fully dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Vasopressin and phenylephrine when added alone had small or negligible effects on the phosphorylation state of the enzyme, as judged from the expressed/total activity ratio. However, when added in combination with glucagon, they elicited appreciable increases in the phosphorylation of the enzyme. Glucagon on its own had no effect either on phosphorylation state or on total HMG-CoA reductase activity during 40 min of incubation. Under conditions of sustained Ca2+ influx (i.e. vasopressin or phenylephrine plus glucagon), there was a marked loss of total HMG-CoA reductase activity. This effect was more pronounced when vasopressin was used; 50% of the enzyme activity was lost within 40 min. The involvement of Ca2+ in these effects was verified directly by the use of ionophore A23187. Its addition to hepatocytes resulted both in a very pronounced increase in the phosphorylation state of the enzyme and in the loss of 50% of the total activity within 30 min. There was no correlation between the ability of any set of conditions to increase the phosphorylation of the enzyme and the subsequent loss of total HMG-CoA reductase activity. The latter parameter appeared to be directly related, however, to the maintenance of prolonged Ca2+ influx, as indicated by the continued activation of glycogen phosphorylase, measured in the same cells. The lack of a causal relationship between increased phosphorylation and loss of total activity was demonstrated directly by studies in which okadaic acid was used to induce phosphorylation of HMG-CoA reductase in hepatocytes by inhibition of phosphatase 1 and 2A activities. This was not accompanied by any loss of total enzyme activity. Neither did okadaic acid enhance the loss of reductase induced by A23187 when the two agents were added together. It is concluded that altered Ca2+ fluxes in hepatocytes in vivo, under conditions of acute or chronic stress (such as may be associated with trauma or diabetes respectively), may be involved in the regulation of the expression of HMG-CoA reductase activity through alteration of enzyme concentration in the liver.
The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin
Authors: Wallin R , Rossi F , Loeser R , Key L L
Published: August 2015

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an ‘in vitro’ system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.
Binding of tissue plasminogen activator to human aortic endothelial cells
Authors: Sanzo M A , Howard S C , Wittwer A J , Cochrane H M
Published: August 2015

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.
Translocation of the brain-type glucose transporter largely accounts for insulin stimulation of glucose transport in BC3H-1 myocytes
Authors: Calderhead D M , Kitagawa K , Lienhard G E , Gould G W
Published: August 2015

Insulin-stimulated glucose transport was examined in BC3H-1 myocytes. Insulin treatment lead to a 2.7 +/- 0.3-fold increase in the rate of deoxyglucose transport and, under the same conditions, a 2.1 +/- 0.1-fold increase in the amount of the brain-type glucose transporter (GLUT 1) at the cell surface. It has been shown that some insulin-responsive tissues express a second, immunologically distinct, transporter, namely GLUT 4. We report here that BC3H-1 myocytes and C2 and G8 myotubes express only GLUT 1; in contrast, rat soleus muscle and heart express 3-4 times higher levels of GLUT 4 than GLUT 1. Thus translocation of GLUT 1 can account for most, if not all, of the insulin stimulation of glucose transport in BC3H-1 myocytes. On the other, hand, neither BC3H-1 myocytes nor the other muscle-cell lines are adequate as models for the study of insulin regulation of glucose transport in muscle tissue.
Cell surface complexes (‘cortices’) isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies
Authors: Lumpert C J , Kersken H , Plattner H
Published: August 2015

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5′-[gamma-thio]triphosphate (ATP[S]) and adenosine 5′[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.
Alkaline serine proteinase from Thermomonospora fusca YX. Stability to heat and denaturants
Authors: Kristjansson M M , Kinsella J E
Published: August 2015

The serine proteinase isolated from Thermomonospora fusca YX shows considerable thermal stability up to 80 degrees C, and progressive inactivation occurs at higher temperatures. Lyotropic salts affected the thermal stability of the enzyme at 85 degrees C, suggesting that disruption of hydrophobic interactions play an important role in the decreased thermal stability of the enzyme above 80 degrees C. Thermal stability is highly pH-dependent; above pH 6.0-6.5 there is a sharp decrease in the stability of the enzyme, reflecting increased autolysis. Although some stabilization occurs upon increasing ionic strength, Ca2+ binding does not appear to play a role in thermal stability. Denaturants, i.e. 8 M-urea, 6 M-guanidinium chloride or 1% SDS, had no significant effect on the activity of the enzyme after 24 h at 25 degrees C.