bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1
Authors: Farrell D H , Cunningham D D
Published: August 2015

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.
Biosynthesis of insulin secretory granule membrane proteins. Control by glucose
Authors: Grimaldi K A , Siddle K , Hutton J C
Published: August 2015

The biosynthesis of a component SGM 110, specifically localized to the membrane of insulin secretory granules, was studied in rat insulinoma cells and in normal islets of Langerhans. Cells or islets were labelled with [35S]methionine or [3H]mannose and SGM 110 was immunoprecipitated by using a monoclonal antibody. Pulse-chase experiments demonstrated that the nascent polypeptide was cotranslationally glycosylated to form a 97,000 Da peptide which in turn was processed to the mature 110,000 Da form. A 50,000 Da form detected by immunoblotting with the same antibody was not conspicuously labelled even after a 20 h chase incubation, suggesting that it represented late processing of SGM 110 in lysosomes. With insulinoma cells, an increase in medium glucose concentration from 3 mM to 20 mM was without effect on the secretion of insulin or on the biosynthesis of (pro)insulin or SGM 110. In normal islets, however, 20 mM-glucose produced a 17-fold increase in (pro)insulin biosynthesis and a 13-fold increase in SGM 110 biosynthesis, compared with only a 2-fold increase in total protein synthesis, as judged by incorporation of [35S]methionine during a 1 h incubation. The effect of glucose on both (pro)insulin and SGM 110 biosynthesis was blocked by the addition of mannoheptulose, but not by the removal of extracellular calcium, both of which conditions inhibit insulin secretion. In contrast tolbutamide, an agent which stimulates insulin secretion, did not enhance the biosynthesis of (pro)insulin or SGM 110. It is concluded that at least one protein component of the insulin secretory granule membrane is synthesized co-ordinately with proinsulin and is subject to similar regulatory mechanisms. Factors which acutely control insulin secretion may also control granule biogenesis, although the two processes are not coupled in an obligatory fashion.
A rapid separation method for inositol phosphates and their isomers
Authors: Wreggett K A , Irvine R F
Published: August 2015

A technique is described using ACCELL QMA anion-exchange SEP-PAKs (Waters Associates) with ammonium formate-based solutions, whereby a sample can be processed within minutes to yield resolution of inositol phosphates. Isomers of inositol trisphosphate can then be separated by using this technique in combination with a rapid (5-6 min) isocratic h.p.l.c. procedure. The use of QMA SEP-PAKs offers a degree of reproducibility comparable with that of h.p.l.c. while maintaining the capacity for automation, allowing large numbers of samples to be processed rapidly.
Extended and globular protein domains in cartilage proteoglycans
Authors: Paulsson M , Mörgelin M , Wiedemann H , Beardmore-Gray M , Dunham D , Hardingham T , Heinegård D , Timpl R , Engel J
Published: August 2015

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3′-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].
Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation
Authors: Santa-Coloma T A , Bley M A , Charreau E H
Published: August 2015

The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.
The identification of epitopic sites in human α1-proteinase inhibitor
Authors: Zhu X J , Kang S S , Hargrove K , Shochat D , Jarrells M , Mojesky M , Chan S K
Published: August 2015

Human α 1-proteinase inhibitor (α 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized α 1-PI and mouse polyclonal antibodies against native α 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between α 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native α 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized α 1-PI as the antigen. The ability of the monoclonal antibodies to bind native α 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding α 1-PI and only fragment I. Antibodies in groups II and III bound α 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between α 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in α 1-PI. Specific monoclonal antibodies to three of these sites were obtained.
Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation
Authors: Ngai P K , Walsh M P
Published: August 2015

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.
The role of topogenic sequences in the movement of proteins through membranes
Authors: Robinson A , Austen B
Published: August 2015

Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table 2). Whereas co-translational translocation and processing were previously considered the norm at the endoplasmic reticulum membrane, several instances of post-translational translocation into endoplasmic reticulum microsomes in vitro have now been described. However, it must be noted that post-translational translocation in vitro is much less efficient than when endoplasmic reticulum membranes are present during translation, and it is possible that in the intact cell translocation occurs during translation. Movement of proteins into chloroplasts and mitochondria occurs after translation. When translocation is post-translational, proteins may perhaps traverse the membrane as folded domains, and the conformational effects of topogenic sequences on these domains may be as envisaged in Wickner's ‘membrane-trigger hypothesis’. Both signal and transit sequences possess amphipathic structures which are capable of interacting with phospholipid bilayers, and these interactions may disturb the bilayer sufficiently to allow entry of the following domains of protein. There is increasing evidence that GTP is required to bind ribosomes and their associated nascent chains to the endoplasmic reticulum membrane. Precisely how the cell's energy is applied to achieve translocation is not clear, but one possibility at the endoplasmic reticulum is that a GTP-hydrolysing transducing mechanism may exist to couple signal sequence receptor binding to movement of the nascent chain across the membrane. Electrochemical gradients are required for protein movement to the mitochondrial inner membrane and across the bacterial inner membrane. Cytoplasmic factors such as SRP, the secA gene product or a 40 kDa protein (for mitochondrial precursors) may act by binding to topogenic sequences and preventing precursor proteins as they are translated from folding into forms which cannot be translocated. Specificity in the cell may be achieved both by targetting interactions between these cytoplasmic factors and their receptors located in target membranes, and also by specific binding of the topogenic sequences to specific proteins integrated into the target membranes. Possible candidates for the latter are the protein of microsomal membranes that reacts with a photoreactive signal peptide to give a 45 kDa complex (Fig. 1), the secY gene product of the bacterial inner membrane, and receptors on the outer membranes of chloroplasts and mitochondria. Whether these aid translocation as well as recognition is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of an NADH-dependent haem-degrading system in ox heart mitochondria
Authors: Kutty R K , Maines M D
Published: August 2015

We report the identification of an NADH-dependent haem-degrading system in ox heart mitochondria. The activity was localized to the mitochondrial inner membrane, specifically associated with complex I (NADH:ubiquinone oxidoreductase). The mitochondrial NADH-dependent haem-degradation activity was highly effective and displayed a rate nearly 60% higher than that of the microsomal activity. The following observations suggested the enzymic nature of the activity: (i) haem degradation by complex I did not proceed upon exposure to elevated temperature and extremes of pH; (ii) it displayed substrate specificity; (iii) it was inhibited by a substrate analogue; and (iv) it showed a cofactor requirement. Moreover, the activity was distinctly different from the ascorbate-mediated haem-degradation activity. Also, complex I differed from the microsomal NADPH:cytochrome c (P-450) reductase inasmuch as the formation of an effective interaction with the microsomal haem oxygenase could not be detected. Addition of purified haem oxygenase to complex I neither influenced the rate of haem degradation nor resulted in the formation of biliverdin IX alpha. In contrast, addition of haem oxygenase to NADPH:cytochrome c (P-450) reductase enhanced the rate of haem degradation by nearly 8-fold, and more than 60% of the degraded haem could be accounted for as biliverdin IX alpha. The haem-degrading activity of complex I appeared to involve the activity of H2O2, as the reaction was inhibited by nearly 90% by catalase, and propentdyopents were detected as reaction products. Intact haemoproteins such as cytochrome c and myoglobin were not effective substrates. However, the haem undecapeptide of cytochrome c was degraded at a rate equal to that observed for haem. Haematohaem was degraded at a rate 50% lower than that observed for haem. It is suggested that the NADH-dependent haem-degradation system may have a biological role in the regulation of the concentration of respiratory haemoproteins and the disposition of the aberrant forms of the mitochondrial haemoproteins.
Clones of human ribosomal DNA containing the complete 18 S-rRNA and 28 S-rRNA genes. Characterization, a detailed map of the human ribosomal transcription unit and diversity among clones
Authors: Maden B E H , Dent C L , Farrell T E , Garde J , McCallum F S , Wakeman J A
Published: August 2015

We have isolated several new clones of human ribosomal DNA. Each clone contains part of the external transcribed spacer, a complete 18 S-rRNA gene, the internal transcribed spacers, a complete 28 S-rRNA gene and a short downstream flanking region. We present a detailed map of the human ribosomal transcription unit with the locations of numerous useful restriction sites. In particular, a unique NheI site in the 5.8 S-rRNA gene enabled this gene to be mapped with respect to the 18 S-rRNA and 28 S-rRNA genes. The human 45 S-rRNA coding region is approx. 13,000 nucleotide residues long, of which the external transcribed spacer comprises approx. 3700 nucleotide residues and the first and second internal transcribed spacers comprise approx. 1070 and 1200 nucleotide residues respectively. A partial survey for sites of variation between clones has revealed a single point of variation among 18 S-rRNA gene sequences (a T/C variation at position 140), several sites of length variation in the regions of the transcribed spacers closely flanking the 18 S-rRNA genes, and some sites of length variation among 28 S-rRNA genes. Most of these sites of variation are associated with simple sequence tracts and are in regions that are known to undergo relatively rapid evolutionary divergence. In particular, the sites of variation among 28 S-rRNA genes occur in G + C-rich tracts whose lengths vary among vertebrates and that can be correlated with extensive hairpin structures previously observed by electron microscopy. Each of the clones so far surveyed in detail differs from the others in one or more respects.
Cystine exodus from normal leucocytes is stimulated by MgATP
Authors: Greene A A , Clark K F , Smith M L , Schneider J A
Published: August 2015

Cystine exodus from partially purified granular fractions of normal leucocytes is stimulated by MgATP. N-Ethylmaleimide, an inhibitor of the lysosomal H+-translocating ATPase, eliminated the stimulated exodus, but had no effect on basal exodus. As the initial content of cystine was increased, the initial velocity of both the basal and ATP-stimulated egress increased. However, as saturation with substrate was approached, the ATP stimulation disappeared leaving only the N-ethylmaleimide-insensitive basal exodus. The increased initial velocity in the presence of ATP may represent improved binding of cystine to the partially saturated inner transporter as a result of conformational or charge optimization brought about by the action of the H+-translocating ATPase.
Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes
Authors: Woods N M , Cuthbertson K S R , Cobbold P H
Published: August 2015

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.
Insulin modifies the properties of glucose transporters in rat brown adipose tissue
Authors: Greco-Perotto R , Assimacopoulos-Jeannet F , Jeanrenaud B
Published: August 2015

The properties of glucose transporters associated with plasma and microsomal membranes have been studied in brown adipose tissue of rats after treatment by saline infusion or hyperinsulinaemic/euglycaemic clamp. In this tissue, insulin produces a 40-fold increase in glucose utilization as measured by the 2-deoxy-D-glucose technique, and therefore a 40-fold increase in the rate-limiting glucose transport. This increase, promoted by insulin, is associated with: (a) translocation of the transporters from a pool associated with the microsomal fraction to the plasma membrane without modification of the total number of transporters; (b) an increase in the Hill coefficient of the plasma-membrane glucose transporters for cytochalasin B from 1.1 to 2.5, indicating the presence of positive co-operativity; (c) a decrease in the Kd (apparent dissociation constant) of the transporters towards cytochalasin B from 148 to 82 nM; (d) no change in the Hill coefficient or Kd for the transporters associated with the microsomal membranes. These data indicate that, in addition to causing translocation of the glucose transporters, insulin modifies their properties and behaviour towards cytochalasin B. This may reflect modifications in their properties and behaviour towards glucose, and by this contribute to bringing about the marked effect of this hormone on glucose transport in brown adipose tissue.
Sex differences in the hydroxylation of cholecalciferol and of 5 β-cholestane-3 α, 7 α, 12 α-triol in rat liver
Authors: Saarem K , Pedersen J I
Published: August 2015

The effect of sex hormones on hydroxylation of cholecalciferol (‘vitamin D3’) and of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol has been investigated in female- and male-rat livers. The mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities were respectively 4.6- and 2.7-fold higher in female- than in male-rat livers. The microsomal 1 alpha-hydroxycholecalciferol 25-hydroxylase was 2.8-fold higher in male- than in female-rat liver. No significant difference was found in the microsomal 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Liver microsomes (microsomal fractions) from male, but not from female, rats also catalysed 1-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. Injection of testosterone into female rats decreased the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities, but not to a statistically significant extent. Testosterone treatment had no effect on the microsomal hydroxylases in female-rat liver. Injection of oestradiol valerate to male rats resulted in increased activities of both mitochondrial hydroxylases to the same levels as those of control females, while the microsomal enzyme activities decreased. The present results indicate that sex hormones exert a regulatory control on the mitochondrial cholecalciferol 25-hydroxylase and C27-steroid 27-hydroxylase activities.
A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells
Authors: Yamada K , Okano Y , Miura K , Nozawa Y
Published: August 2015

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.
Studies of the decrease of tyrosine-O-sulphated proteins in Rous sarcoma-virus-transformed rat embryo fibroblasts, line 3Y1. Examination of the sulphate activation and tyrosyl-protein sulphotransferase systems
Authors: Liu M C , Suiko M , Lipmann F
Published: August 2015

The sulphate activation and tyrosyl-protein sulphotransferase systems in normal 3Y1 rat embryo fibroblasts and the same cells transformed by Schmidt Ruppin subgroup-A-Rous sarcoma virus (SRA-3Y1) were examined. Employing metabolic [35S]sulphate-labelling followed by PEI (polyethyleneimine)-cellulose thin-layer chromatography of the labelled cell lysates, it was found that the steady-state level of ‘active’ sulphate, adenosine 3′-phosphate 5′-phosphosulphate, was drastically lower in SRA-3Y1 cells compared with their normal counterparts. When the sulphate activating enzymes were tested, it appeared that the activities in 3Y1 homogenates were 2-2.5 times greater than those in SRA-3Y1 homogenates. An endogenous sulphation assay for tyrosyl-protein sulphotransferase revealed that activities in 3Y1 and SRA-3Y1 homogenates were comparable. Nearly identical patterns were observed with both sets of cells when [35S]sulphated proteins generated in the endogenous assay were separated by two-dimensional gel electrophoresis. It therefore seems that the tyrosyl-protein sulphotransferase(s) are unimpaired in SRA-3Y1 cells. While the lower (approx. 8 times) sulphate uptake remains the major cause for the decrease of tyrosine-O-sulphated proteins in SRA-3Y1 cells [Liu & Lipmann, (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3695-3698], the 2-2.5-fold lower sulphate activating enzyme activities also contribute to some extent to the difference between the SRA-3Y1 and 3Y1 cells.
Quantitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine adrenal glomerulosa cells. Different requirements for angiotensin II and K+
Authors: Capponi A M , Lew P D , Vallotton M B
Published: August 2015

Angiotensin II (AII) and K+ raise the cytosolic free Ca2+ concentration [(Ca2+]i) and stimulate aldosterone production in isolated bovine adrenal glomerulosa cells. The mechanisms leading to an elevation of [Ca2+]i were analysed with the fluorescent Ca2+ probe quin 2. (1) Whereas [Ca2+]i rose transiently and returned to basal values within 5 min in response to AII, the effect of K+ was sustained for at least 15 min. (2) AII released Ca2+ from intracellular stores, whereas the [Ca2+]i response to K+ depended solely on extracellular [Ca2+]. (3) When added after K+ stimulation, AII provoked a dramatic decrease in [Ca2+]i to below the resting value. The role of [Ca2+]i in stimulating steroidogenesis was determined by manipulating the concentration of this cation. (4) In a cell superfusion system, the aldosterone response to AII is biphasic. Suppressing the transient [Ca2+]i elevation triggered by AII resulted in the disappearance of the initial secretory peak, but the final production rate was similar to that of control cells. (5) Normal basal [Ca2+]i levels were, however, necessary to maintain continuous AII-induced steroidogenesis. (6) When added after AII, the antagonist analogue [Sar1,Ala8]AII suppressed steroidogenesis without affecting [Ca2+]i levels. (7) In contrast, continuously elevated [Ca2+]i values were required for the initiation and the maintenance of K+-stimulated aldosterone production. These results demonstrate important differences in the mechanisms through which AII and K+ activate the Ca2+ messenger system. Moreover, functional correlations have shown that K+, but not AII, depends solely on a sustained [Ca2+]i response for its steroidogenic effect. However, the AII-induced effect is also a Ca2+-requiring process: the initial [Ca2+]i transient accelerates the onset of steroidogenesis, which is subsequently extremely sensitive to [Ca2+]i decreases below normal basal levels.
The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae
Authors: Dickinson F M , Haywood G W
Published: August 2015

The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was studied. An enzyme group with pKa approx. 7.1 apparently promotes enzyme activity when in its alkaline form.
Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation
Authors: Watanabe T K , Kuczmarski E R , Reddy J K
Published: August 2015

Myosin has been identified in a variety of non-muscle cells, and is believed to play a role in maintenance of cell shape, locomotion, cytokinesis, exocytosis and other cellular functions. In this paper we describe the purification of myosin from a pancreatic acinar-cell carcinoma of the rat which forms solid tumours, but retains many differentiated functions. The purified myosin was composed of a 200,000 Da heavy chain and two or three classes of light chains. Electron-microscopic examination of rotary-shadowed preparations revealed that individual molecules had two globular heads and a long tail measuring approx. 149 nm. The myosin was soluble in high-salt buffers and became sedimentable as the ionic strength was lowered. Examination of negative-stained preparations showed that this sedimentable myosin consisted of short, bipolar, thick filaments which had a strong tendency to aggregate in a head-to-head manner. The ATPase activity of the purified myosin was stimulated by EDTA or Ca2+, but not by Mg2+. In low ionic strength the Mg2+-dependent ATPase activity was activated by muscle f-actin. The pancreatic myosin bound to actin and could be dissociated by the addition of MgATP. Myosin purified from cells cultured in media containing [32P]Pi was phosphorylated on one of the light chains as well as the heavy chain. Thus pancreatic acinar cells contain a typical non-muscle myosin, and the subunits of this molecule are subject to post-translational modification by phosphorylation.
Anion transport in basolateral (sinusoidal) liver plasma-membrane vesicles of the little skate (Raja erinacea)
Authors: Hugentobler G , Fricker G , Boyer J L , Meier P J
Published: August 2015

The mechanism(s) of [35S]sulphate transport was investigated in basolateral liver plasma-membrane vesicles of the little skate elasmobranch, Raja erinacea. Imposition of an intravesicular alkaline pH gradient (pH 8.0 in/pH 6.0 out) stimulated sulphate uptake 5-10-fold compared with pH-equilibrated (pH 8.0 in = out) conditions and 2-3-fold over equilibrium sulphate uptake (overshoot). This pH-gradient-stimulated sulphate uptake was temperature-dependent, saturable with increasing concentrations of sulphate and could be inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and the anion-transport inhibitors 4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid (DIDS) and probenecid, cis-Inhibition of pH-gradient-driven sulphate uptake was observed with sulphate, oxalate, cholate and bromosulphophthalein, but not with chloride and taurocholate. In addition, sulphate and oxalate trans-stimulated [35S]sulphate uptake under pH-equilibrated conditions. Although also stimulated by an inside-alkaline pH gradient, transmembrane transport of [3H]cholate was not inhibited by DIDS, suggesting that its pH-gradient-driven uptake is not mediated by an anion-transport ‘carrier’. In conclusion, these studies indicate that a basolateral plasma-membrane sulphate-transport system has evolved in skate hepatocytes and is similar to that in mammalian liver cells. This archaic anion-exchange system co-transports certain organic anions such as oxalate and has developed early in vertebrate evolution.
Phosphatidic acid and arachidonic acid each interact synergistically with glucagon to stimulate Ca2+ influx in the perfused rat liver
Authors: Altin J G , Bygrave F L
Published: August 2015

The administration of phosphatidic acid to rat livers perfused with media containing either 1.3 mM- or 10 microM-Ca2+ was followed by a stimulation of Ca2+ efflux, O2 uptake and glucose output. The responses elicited by 100 microM-phosphatidic acid were similar to those induced by the alpha-adrenergic agonist phenylephrine. Contrary to suggestions that phosphatidic acid acts like a Ca2+-ionophore, no net influx of Ca2+ was detected until the phosphatidic acid was removed. Sequential infusions of phenylephrine and phosphatidic acid indicate that the two agents release Ca2+ from the same intracellular source. The co-administration of glucagon (or cyclic AMP) and phosphatidic acid, and also of glucagon and arachidonic acid, led to a synergistic stimulation of Ca2+ uptake of the liver, a feature similar to that observed after the co-administration of glucagon and other Ca2+-mobilizing hormones [Altin & Bygrave (1986) Biochem. J. 238, 653-661]. A notable difference, however, is that the synergistic stimulation of Ca2+ uptake induced by the co-administration of glucagon and arachidonic acid was inhibited by indomethacin, whereas that induced by glucagon and phosphatidic acid, or glucagon and other Ca2+-mobilizing agents, was not. The results suggest that the synergistic action of glucagon and arachidonic acid in stimulating Ca2+ influx is mediated by prostanoids, but that of glucagon and phosphatidic acid is evoked by a mechanism similar to that of Ca2+-mobilizing agents.
Proto-oncogene expression in proliferating and differentiating cardiac and skeletal muscle
Authors: Claycomb W C , Lanson N A
Published: August 2015

We have examined the expression of 13 proto-oncogenes in proliferating and terminally differentiated cardiac and skeletal muscle. Total RNA was prepared from intact ventricular cardiac-muscle tissue and from purified ventricular cardiac-muscle cells of neonatal and adult rats and from cultured proliferating and terminally differentiated L6A1 rat skeletal-muscle cells. cDNA probes for histone H4, thymidine kinase, myosin heavy chain and M-creatine kinase were used to assess cellular proliferation and differentiation. Oncogenes c-myc, c-raf, c-erb-A, c-ras-H, c-ski, and c-sis were expressed in both proliferating and differentiated cardiac muscle tissue and cells, whereas c-myb expression was not observed in either. c-src was expressed only in neonatal cardiac muscle tissue and cells. c-fms, c-abl, and c-ras-K were expressed in tissue from both neonatal and adult animals but only in purified cells from neonatal animals. c-fes/fps was expressed only in neonatal cardiac muscles cells. c-fos expression was not observed in cardiac-muscle tissue from either neonatal or adult rats, but surprisingly was abundantly expressed in freshly isolated cardiac-muscle cells from animals of both ages. These results emphasize that biochemical analysis using intact cardiac-muscle tissue may not necessarily reflect muscle-specific cell processes. They also show that the expression of c-fos can be activated by the cell isolation procedure. c-myc, c-ski, c-ras-H, c-ras-K, c-abl, c-raf and c-erb-A were expressed in both proliferating and terminally differentiated skeletal-muscle cells, whereas c-myb, c-fos, c-src and c-fms transcripts were observed only in proliferating cells. c-fes/fps and c-sis were not expressed in dividing or fused skeletal-muscle cells. These results demonstrate unique tissue and cell-specific patterns of proto-oncogene expression and suggest that these genes may be involved with the regulation of cellular proliferation and terminal differentiation in striated muscle.
Continued glucose output after re-feeding contributes to glucose intolerance in hyperthyroidism
Authors: Holness M J , Sugden M C
Published: August 2015

The effects of hyperthyroidism to elicit glucose intolerance after glucose administration were decreased under conditions where hepatic glucose output was suppressed. It is concluded that continued hepatic glucose output contributes to abnormal glucose tolerance in hyperthyroidism.
Characterization of three peptides derived from prosomatostatin [prosomatostatin-(1–63)-, -(65–76)- and -(79–92)-peptides] in a human pancreatic tumour
Authors: Conlon J M , Eriksson B , Grimelius L , Oberg K , Thim L
Published: August 2015

By using only reverse-phase h.p.l.c., three fragments of prosomatostatin were isolated from an extract of a human pancreatic neuroendocrine tumour that produced somatostatin, vasoactive intestinal polypeptide and gastrin-releasing peptide. The amino acid composition of the peptides indicated that they represented prosomatostatin-(1-63)-peptide, prosomatostain-(65-76)-peptide and prosomatostatin-(79-92)-peptide (somatostatin-14). The identity of prosomatostatin-(1-63)-peptide was confirmed by characterization of the products of digestion with Armillaria mellea (honey fungus) proteinase. Partial micro-sequencing of prosomatostatin-(1-63)-peptide showed that the Gly24-Ala25 bond of preprosomatostatin was the site of cleavage of the signal peptide. Thus human prosomatostatin is a protein of 92 amino acid residues that is proteolytically cleaved in a pancreatic tumour at the site of a dibasic-residue (arginine-lysine) processing site and at a single-monobasic-residue (arginine) processing site.
H.p.l.c. analysis of inositol monophosphate isomers formed on angiotensin II stimulation of rat adrenal glomerulosa cells
Authors: Bird I M , Smith A D , Schulster D
Published: August 2015

[3H]Inositol-prelabelled isolated rat adrenal glomerulosa cells were stimulated with 25 nM-AII ([Asp1, Ile5]-angiotensin II) in the presence of 10 mM-Li+, and the resulting inositol monophosphate isomers were separated successfully by using a recently developed h.p.l.c. methodology. Two major peaks of radioactivity were detected which showed the same retention characteristics on h.p.l.c. as inositol 4-phosphate and inositol 1-phosphate and which increased 5-fold and 8-fold respectively on stimulation with AII. In addition, a relatively small peak with the retention characteristics of inositol 1:2-cyclic phosphate was seen to undergo a 1.5-fold increase on stimulation. This was not considered sufficient to suggest that cyclic phosphoinositols were a major product of AII-stimulated phosphoinositide turnover. No peaks of radioactive material were detected in the regions expected for inositol 2-phosphate (an acid hydrolysis product of inositol 1:2-cyclic phosphate) or inositol 5-phosphate. These results establish the identity of the major inositol phosphate products in AII-stimulated glomerulosa cells and confirm and extend the previous observations of Balla, Baukal, Guillemette, Morgan & Catt [(1986) Proc. Natl. Acad. Sci. 83, 9323-9327].
Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450
Authors: Shedlofsky S I , Sinclair P R , Bonkovsky H L , Healey J F , Swim A T , Robinson J M
Published: August 2015

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.
Characterization of the inositol 1,4,5-trisphosphate-induced Ca2+ release in pancreatic β-cells
Authors: Nilsson T , Arkhammar P , Hallberg A , Hellman B , Berggren P O
Published: August 2015

Pancreatic beta-cells isolated from obese-hyperglycaemic mice released intracellular Ca2+ in response to carbamoylcholine, an effect dependent on the presence of glucose. The effective Ca2+ concentration reached was sufficient to evoke a transient release of insulin. When the cells were deficient in Ca2+, the Ca2+ pool sensitive to carbamoylcholine stimulation was equivalent to that released by ionomycin. Unlike intact cells, cells permeabilized by high-voltage discharges failed to generate either inositol 1,4,5-triphosphate (InsP3) or to release Ca2+ after exposure to carbamoylcholine. However, the permeabilized cells released insulin sigmoidally in response to increasing concentrations of Ca2+. Also in the absence of functional mitochondria these cells exhibited a large ATP-dependent buffering of Ca2+, enabling the maintenance of an ambient Ca2+ concentration corresponding to about 150 nM even after several additional pulses of Ca2+. InsP3, maximally effective at 6 microM, promoted a rapid and pronounced release of Ca2+. The InsP3-sensitive Ca2+ pool was rapidly filled and lost its Ca2+ late after ATP depletion. The transient nature of the Ca2+ signal was not overcome by repetitive additions of InsP3. It was possible to restore the response to InsP3 after a delay of approx. 20 min, an effect which had less latency after the addition of Ca2+. These latter findings argue against degradation and/or desensitization as factors responsible for the transiency in InsP3 response. It is suggested that Ca2+ released by InsP3 is taken up by a part of the endoplasmic reticulum (ER) not sensitive to InsP3. On metabolism of InsP3, Ca2+ recycles to the InsP3-sensitive pool, implying that this pool indeed has a very high affinity for the ion. The presence of functional mitochondria did not interfere with the recycling process. The ER in pancreatic beta-cells is of major importance in buffering Ca2+, but InsP3 only modulates Ca2+ transport for a restricted period of time following immediately upon its formation. Thereafter the non-sensitive part of the ER takes over the continuous regulation of Ca2+ cycling.
Purification, characterization and proteinase-inhibitory activity of a Clara-cell secretory protein from the pulmonary extracellular lining of rabbits
Authors: Gupta R P , Patton S E , Jetten A M , Hook G E R
Published: August 2015

A low-Mr Clara-cell secretory protein, CCSP, previously shown to be a major secretory product of Clara cells, was isolated from rabbit lung lavage effluents. CCSP accounted for 4.4 +/- 0.5% of the protein in the soluble phase of cell- and surfactant-free pulmonary lavage effluents (LE). Purification of this protein from LE was achieved in two steps. First, the LE was acidified with HCIO4 and, secondly, CCSP was isolated by gel-exclusion chromatography on Sephadex G-50. Purified CCSP was homogeneous by SDS/polyacrylamide-gel electrophoresis (PAGE), consisting of a single major isoform with a pI of 6.0. The Mr of CCSP was 5800 according to SDS/PAGE under reducing conditions and 12,600 under non-reducing conditions. However, by gel chromatography the Mr of the protein under non-reducing conditions was 12,400 and under reducing conditions it increased to 15,000. The discrepancy obtained by using these two techniques was attributed to anomalous electrophoretic mobilities of the protein in its reduced state. The molecule contained three half-cystine residues, but no free thiol groups, and tryptophan was not detectable. The first seven N-terminal amino acid residues were Gly-Ile-Xaa-Pro-Arg-Phe-Ala-. The third residue was not identified. CCSP showed inhibitory activity against the thiol proteinase papain (50% inhibition at 4 microM-CCSP), but only weak activities against human polymorphonuclear-leucocyte elastase, and bovine trypsin. The molecule was not digested by, and did not complex with, trypsin. CCSP was immunochemically different from surfactant apoprotein B (Mr 10,000) present in rabbit lung surfactant. This study is the first partial characterization of the major secretory protein of rabbit lung Clara cells.
Structural and compositional differences between intracellular and secreted mucin of rat small intestine
Authors: Fahim R E F , Forstner G G , Forstner J F
Published: August 2015

An investigation was undertaken to discover whether mucin purified from secretions in the lumen of rat small intestine differed in structure or composition from intracellular mucin purified from rat intestinal tissue. To do this, ligated loops were constructed in situ from previously washed intestinal segments and mucin purified separately from tissue homogenates or loop fluid. Secreted mucin (SM) differed from intracellular mucin (IM) by having a higher proportion of ‘minor’ mucin amino acids (aspartic acid, glutamic acid, glycine and alanine) and a lower proportion of ‘major’ amino acids (serine, proline and threonine). SM also contained less N-acetylgalactosamine and a small, but measureable, amount of mannose. Gel electrophoresis showed that SM penetrated the gel more readily and, unlike IM, gave a rather prominent, but diffuse, band having a midpoint position of Mr 200,000. After reduction both IM and SM gave rise to the putative ‘link’ component of Mr 118,000 and the 200,000-Mr band of SM disappeared. SM was included to a greater extent than IM on Sepharose CL-2B chromatography, suggesting a smaller size. With the use of CsCl-density-gradient ultracentrifugation of SM, a lighter species [buoyant density (rho) = 1.38 g/ml] enriched in the 200,000-Mr component, was separated from a heavier, more glycosylated, species (rho = 1.50 g/ml). Purified 200,000-Mr component had a composition identical with that of the 118,000-Mr ‘link’ component of IM, reacted in Western blots with an antibody specific for the 118,000-Mr ‘link’ component, and after reduction gave rise to a 118,000-Mr component on gel electrophoresis. Thus secreted mucin contains a 200,000-Mr component which appears to represent a disulphide-linked dimer of the previously described 118,000-Mr ‘link’ component of intracellular mucin.
Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A
Authors: Vamecq J , Schepers L , Parmentier G , Mannaerts G P
Published: August 2015

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.