bluuebunny/crossref_metadata_2025 huggingface dataset:
The lung lectin surfactant protein A aggregates phospholipid vesicles via a novel mechanism
Authors: Haagsman H P , Elfring R H , van Buel B L M , Voorhout W F
Published: August 2015

Surfactant protein A (SP-A), a lung-specific glycoprotein, consists of an N-terminal collagen-like domain and a C-terminal domain with a sequence similar to that of several Ca2(+)-dependent lectins. SP-A induces a rapid Ca2(+)-dependent aggregation of phospholipid vesicles. We report here that vesicle aggregation is mediated by Ca2(+)-induced interactions between carbohydrate-binding domains and oligosaccharide moieties of SP-A. This novel mechanism of membrane interactions may be relevant to the formation of the membrane lattice of tubular myelin, an extracellular form of surfactant.
Cyclic AMP-evoked oscillations of intracellular [Ca2+] in guinea-pig hepatocytes
Authors: Capiod T , Noel J , Combettes L , Claret M
Published: August 2015

The effects of the beta-adrenoceptor agonist isoprenaline and cyclic AMP (cAMP) on cytosolic free Ca2+ ([Ca2+]i) were studied in the single guinea-pig hepatocyte. In common with InsP3-dependent agonists such as noradrenaline or angiotensin II, isoprenaline (0.5-10 microM) and cAMP (50-100 mM, perfused into the cell via the patch-pipette), were able to generate fast and slow fluctuations of [Ca2+]i. Responses to isoprenaline and cAMP also were observed in the absence of external Ca2+. Isoprenaline-evoked [Ca2+]i rises were not blocked by the intracellular perfusion of heparin, suggesting that these fluctuations are independent of the binding of InsP3 to its receptor.
Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase
Authors: Hawkins H C , de Nardi M , Freedman R B
Published: August 2015

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42 microM at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5. This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.
Thrombin induces a biphasic 1,2-diacylglycerol production in human platelets
Authors: Nakashima S , Suganuma A , Matsui A , Nozawa Y
Published: August 2015

The 1,2-diacylglycerol (DAG) mass content was measured in thrombin-stimulated human platelets. Thrombin stimulates a biphasic accumulation of DAG, with an early phase reaching a peak at 10 s and a later phase reaching a peak at 2-3 min. The time course of first-phase DAG production corresponded well to that of Ins(1,4,5)P3 formation, which was rapid and transient. The second phase of DAG accumulation occurred after the level of Ins(1,4,5)P3 returned to nearly basal. Thrombin stimulated the decrease in PtdIns and phosphatidylcholine contents. The source of second-phase DAG was examined in platelets prelabelled with three radioactive fatty acids, i.e. arachidonic, palmitic and myristic. Thrombin stimulated the increase in radioactivity of DAG with decline of PtdIns in platelets labelled with [3H]arachidonic acid or [3H]palmitic acid, in which PtdIns was considerably labelled. In contrast, significant accumulation of [3H]DAG was not observed in [3H]myristic acid-labelled platelets, in which PtdIns was poorly labelled. In platelets prelabelled with [3H]inositol, an increase in InsP in response to thrombin was seen for more than 5 min. In contrast, upon stimulation, significant increases in [3H]phosphocholine and [3H]choline were not observed in [methyl-3H]choline-labelled platelets. Thrombin induced a small production of phosphatidylethanol, when ethanol was present during stimulation. However, the formation of DAG and phosphatidic acid was not significantly affected by ethanol. These results suggest that thrombin stimulates a biphasic accumulation of DAG, initially from PtdInsP2 and later from PtdIns in human platelets.
Inhibition of luteinizing-hormone exocytosis by guanosine 5′-[γ-thio]triphosphate reveals involvement of a GTP-binding protein distal to second-messenger generation
Authors: van der Merwe P A , Millar R P , Wakefield I K , Davidson J S
Published: August 2015

Dual inhibitory and stimulatory actions of guanine nucleotides on luteinizing-hormone (LH) exocytosis were observed in primary sheep gonadotropes permeabilized with staphylococcal alpha-toxin. At resting cytosolic [Ca2+]free (pCa 7), 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated rapid LH exocytosis, which was maximal between 5 and 10 min. GTP[S] and p[NH]ppG had similar potencies (50% of maximum effect at 20-50 microM), but the effect of p[NH]ppG was more prolonged. Experiments carried out in the presence of saturating concentrations of phorbol 12-myristate 13-acetate (PMA), or in PMA-desensitized cells, suggested that stimulation by p[NH]ppG is mediated by a mechanism additional to protein kinase C (PKC) activation. Furthermore, p[NH]ppG stimulated LH exocytosis in the presence of saturating cyclic AMP (cAMP) concentrations, although its effect was less than additive. However, when both PMA and cAMP were present, p[NH]ppG did not stimulate a further increase in the rate of LH exocytosis. In contrast, pretreatment of cells with GTP[S] at low [Ca2+]free markedly inhibited subsequent responses to Ca2+, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTP[S] concentrations than the stimulatory effect (50% inhibition at 1-10 microM), and was not observed with p[NH]ppG. A similar inhibition was observed with adenosine 5′-[gamma-thio]triphosphate, probably by its conversion into GTP[S]. These results suggest that the stimulatory actions of guanine nucleotides can be accounted for by the combined activation of PKC and generation of cAMP, resulting from activation of conventional signal-transducing GTP-binding proteins. The inhibitory effect of GTP[S] can be clearly distinguished and indicates the involvement of a distinct GTP-binding protein in exocytosis at a site distal to second-messenger generation.
Immunological studies on the rat peripheral-type benzodiazepine acceptor
Authors: Moynagh P N , Bailey C J , Boyce S J , Williams D C
Published: August 2015

Photoaffinity labelling of rat adrenal mitochondrial preparations with [3H]PK 14105 resulted in a single 3H-labelled band on SDS/PAGE gels with an apparent-molecular-mass peak of 18 kDa. This represents a polypeptide associated with the peripheral-type benzodiazepine-binding site. Solubilization of photoaffinity-labelled membranes with 6 M-guanidine hydrochloride, followed by gel filtration and reversed-phase h.p.l.c. of the solubilized material, resulted in the purification to homogeneity of the [3H]PK 14105-labelled polypeptide. This purified polypeptide was used to raise a rabbit polyclonal antiserum which recognized the immunogen in pure form and exclusively recognized it in a crude preparation of rat adrenal mitochondria as judged by immunoblotting. By the same analysis the antiserum identified the corresponding polypeptide from rat kidney and salivary gland, demonstrating its cross-reactivity. Subsequent immunocytochemical studies localized the polypeptide to the cortex of the adrenal gland, the distal tubules of kidney, the interstitial cells of testis, the biliary epithelium of liver and the choroid plexus and ependyma cells within the brain. This selective localization within organs may provide an insight into the physiological role of the peripheral-type benzodiazepine acceptor.
Synthesis of the acid-labile subunit of the growth-hormone-dependent insulin-like-growth-factor-binding protein complex by rat hepatocytes in culture
Authors: Scott C D , Baxter R C
Published: August 2015

Insulin-like growth factors (IGFs) circulate predominantly in a growth-hormone-dependent ternary complex of 125-150 kDa. This study investigates the production of the alpha-subunit of this complex, an acid-labile glycoprotein without intrinsic IGF-binding activity, which binds to the IGF-binding protein IGFBP-3 in the presence of IGFs. Medium conditioned by primary cultures of rat hepatocytes produced alpha-subunit with similar complex-forming activity to purified rat serum alpha-subunit. Bovine growth hormone stimulated hepatocyte production of both IGF-I and alpha-subunit. IGF-I tracer bound to pure rat IGFBP-3 was converted from approx. 60 kDa to 150 kDa by serum alpha-subunit, whole rat serum or rat hepatocyte culture medium; this converting activity was destroyed by transient acidification. In contrast, IGF-I bound to hepatocyte-medium IGF-binding proteins could not be converted into a high-molecular-mass from by purified rat serum alpha-subunit. Rat serum and hepatocyte-medium alpha-subunit appeared identical by electrophoretic analysis, since reaction of either with cross-linked IGF-I.IGFBP-3 tracer resulted in bands of molecular mass 130 kDa and 160 kDa, probably representing intact and partially deglycosylated complexes. However, IGF-binding proteins in rat serum and hepatocyte medium were different, in that affinity labelling of medium binding proteins, depleted of endogenous IGFs, showed no evidence of the 50-60 kDa cluster of bands characteristic of rat serum IGFBP-3. We conclude that rat hepatocytes in primary culture produce alpha-subunit similar to that in rat serum; however, alpha-subunit is unable to form ternary complexes with hepatocyte IGF-binding proteins, since cultured hepatocytes do not secrete IGFBP-3.
Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding
Authors: Lander M , Pitt A R , Alefounder P R , Bardy D , Abell C , Battersby A R
Published: August 2015

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.
Purification of a high-affinity inositol 1,3,4,5-tetrakisphosphate receptor from brain
Authors: Donié F , Reiser G
Published: August 2015

Ins(1,3,4,5)P4 has been suggested to be involved in cellular Ca2+ signalling. A receptor from pig cerebellar membranes, which binds InsP4 with high affinity and selectivity [Donié & Reiser (1989) FEBS Lett. 254, 155-158], has been solubilized and purified about 20,000-fold by chromatography using CM-cellulose, heparin-agarose and hydroxyapatite. The InsP4 receptor, identified by SDS/PAGE, had an apparent molecular mass of 42 kDa and bound 4.6 nmol of InsP4 per mg of protein, with a dissociation constant of 5.6 nM.
myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate
Authors: Stephens L R , Hawkins P T , Stanley A F , Moore T , Poyner D R , Morris P J , Hanley M R , Kay R R , Irvine R F
Published: August 2015

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate, 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346, 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).
Purification and characterization of truncated ribonuclease inhibitor
Authors: Hofsteenge J , Vincentini A , Stone S R
Published: August 2015

A recombinant pig ribonuclease inhibitor (delta r-RI) lacking 90 or 93 N-terminal amino acid residues was isolated from a preparation of recombinant inhibitor. The kinetic parameters for the inhibition of ribonuclease A by delta r-RI were determined and found to be only slightly altered in comparison with the full-length inhibitor. The deletion did, however, affect the surface properties of RI. The results are discussed in relation to those obtained by Lee & Vallee [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883].
Distinct structural specificities for functional coupling of the epidermal growth factor receptor to calcium-signalling versus phospholipase A2 responses
Authors: Hack N , Margolis B L , Ullrich A , Schlessinger J , Skorecki K L
Published: August 2015

Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca2+, and of phospholipase A2 (PLA2) leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA2. We now demonstrate that coupling of the EGF receptor to PLA2 was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA2 was demonstrated using three different experimental designs: (1) release of [14C]arachidonic acid from prelabelled intact cells. (2) release of [3H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA2 enzymic activity in cell-free extracts using an ‘in vitro’ assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA2 occurred despite the absence of a demonstrable Ca(2+)-signalling response and the detection of diminished but persistent PLC-gamma phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA2 activation versus Ca2+ signalling.
Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission
Authors: Reed C J , Douglas K T
Published: August 2015

In the presence of Cu(II) ions, supercoiled DNA is cleaved in neutral solution by low concentrations of thiols. Supercoiled plasmid DNA is cleaved first to open circular DNA, which in turn produces linear DNA and eventually fragments. Cleavage is strongly temperature-dependent and is maximal at 0.10-0.25 M-NaCl concentration. In the presence of excess of either component of the Cu(II)-thiol pair, the extent of cleavage depended on the concentration of the limiting partner, and was easily detectable down to micromolar concentrations of limiting GSH. Scavengers of oxygen-derived species (such as hydrogen peroxide, superoxide radical ion and hydroxyl radical) indicated that the hydroxyl radical may be involved in the cleavage mechanism. DNA cleavage leads to some production of 2-thiobarbituric acid-reactive species and some of the cleavage sites, at least, had 5′-hydroxy and/or 3′-hydroxy groups. There was extensive base damage before cleavage. Studies with S1 nuclease indicated no gross sequence preference for Cu(II)-GSH cleavage of pSP64 plasmid DNA. The Cu(II)-thiol system did not appear to target special structural features in the DNA such as Z-DNA inserts, cruciform structures or left-handed (but non-Z) DNA. Cleavage might arise from a reagent generated either by the Cu(II)-thiol combination in free solution or by attack involving Cu(II) ions pre-bound to DNA. The attack of GSH plus Cu(II) ions on DNA may be a potential toxic lesion under physiological conditions unless special protective measures operate efficiently in the cell.
Isolation and characterization of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid from human urine and preparation of its proposed precursor, S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine
Authors: Kinuta M , Yao K , Masuoka N , Ohta J , Teraoka T , Ubuka T
Published: August 2015

3-[(Carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid (I) was isolated from healthy human urine by using ion-exchange column chromatography, and characterized by physicochemical analyses involving i.r., m.s. and n.m.r. spectrometries as well as chemical synthesis. The urinary content was 0.04-0.07 mumol/l. Compound (I) was synthesized by the addition of mercaptoacetic acid to urocanic acid. In order to establish the origin of the compound. S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (II) and S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (III) were produced by similar reactions of urocanic acid with cysteine and GSH respectively. The yield of compound (II) was markedly increased by sunlight irradiation of the reaction mixture or by the use of cis-urocanic acid rather than the trans isomer. Incubation of compound (II) with rat liver homogenate in a phosphate buffer, pH 7.40, formed a major and some minor products of enzymic degradation, one of which was identified with compound (I). Exposure of rats to the sunlight for 2 days resulted in increase of the epidermal content of trans-urocanic acid from the normal value of 0.38 to 1.70 micrograms/mg wet wt. of skin, accompanied by formation de novo of the epidermal cis isomer. After sunlight irradiation, the content of the trans isomer decreased at a constant rate of 0.03 micrograms/mg wet wt. of skin per day, whereas the cis isomer was eliminated more quickly, having a phase of rapid decrease in the early period. From these results we suggest that compound (I) may participate in the metabolism of urocanic acid and natural thiol compounds such as cysteine and GSH.
Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues
Authors: Regazzi R , Ullrich S , Kahn R A , Wollheim C B
Published: August 2015

Low-molecular-mass GTP-binding proteins of the ras family were analysed by [32P]GTP binding after PAGE and transfer to nitrocellulose membranes. By this technique, several GTP-binding proteins in the 20-30 kDa range were detected in both cytosolic and microsomal fractions of RINm5F cells. One of these, displaying an apparent molecular mass of about 20 kDa and a pI of 6.7, was mainly cytosolic and was shown to be the ADP-ribosylation factor (ARF) by using specific antibodies. When permeabilized RINm5F cells were incubated with the stable GTP analogues guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) the amount of ARF increased in a fraction containing both Golgi and plasma-membrane markers, but not in the fraction containing secretory granules, mitochondria and lysosomes. GTP, GDP and its beta-thio analogue did not enhance ARF binding to membranes, smg25/rab3 and rho, as well as all the other small GTP-binding proteins detected by the [32P]GTP binding, did not redistribute under these conditions. As GTP[S] stimulates insulin secretion in these cells, we also examined the relationship between ARF translocation and insulin secretion. Both phenomena were elicited by GTP[S] with an EC50 (median effective concentration) of about 10 microM. p[NH]ppG was equipotent with GTP[S] in inducing insulin secretion (EC50 about 10 microM), but higher concentrations (about 500 microns) were required to achieve the same maximal ARF redistribution. These results suggest that: (1) ARF is subject to cycling between a membrane-associated and a free/loosely attached form, determined by the species of bound guanine nucleotide; (2) ARF alone does not seem to regulate exocytosis in insulin-secreting cells.
Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity
Authors: Ramsay R R , Mancinelli G , Arduini A
Published: August 2015

Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.
Similarity between physicochemical properties of recombinant rat prorenin and native inactive renin
Authors: Hosoi M , Kim S , Yamauchi T , Watanabe T , Murakami K , Suzuki F , Takahashi A , Nakamura Y , Yamamoto K
Published: August 2015

Rat prorenin was synthesized by Chinese-hamster ovary cells transfected with an expression vector containing rat preprorenin cDNA sequences, then purified by concanavalin A-Sepharose chromatography and h.p.l.c. on G3000SW. The molecular mass of purified prorenin was 46,000 Da, as determined by h.p.l.c. on G3000SW. Immunoblot analysis indicated that recombinant prorenin cross-reacted with anti-(mature renin) antibody and two kinds of antibodies recognizing the N-terminus and C-terminus of the prosegment of rat prorenin. Recombinant prorenin was bound to a Cibacron Blue-Sepharose column and eluted with 1.4 M-NaCl, but was not retained by an octapeptide renin inhibitor (H-77)-Sepharose column. Trypsin activation of prorenin increased the renin activity 110-fold, caused binding to an H-77-Sepharose column and nullified the reactivity to the above two kinds of anti-prosegment antibodies, findings indicating that the activation of prorenin with trypsin is due to the cleavage of the prosegment. Rat plasma inactive renin, partially purified by h.p.l.c. on G3000SW, had much the same physicochemical characteristics as the recombinant prorenin. These results provide evidence that rat plasma inactive renin is prorenin. Recombinant prorenin is a useful material for examining the physiological role of circulating prorenin.
Ionic interactions in the formation of the thrombin-hirudin complex
Authors: Betz A , Hofsteenge J , Stone S R
Published: August 2015

Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with alpha-thrombin. The contribution to binding energy of acidic residues in the 11 C-terminal amino acids varied from 2.3 to 5.9 kJ.mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ.mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue. The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.
Liver glucose-6-phosphatase activity is not modulated by physiological intracellular Ca2+ concentrations
Authors: Fulceri R , Bellomo G , Gamberucci A , Benedetti A
Published: August 2015

1. In the presence of MgATP and increasing amounts of added Ca2+, isolated liver microsomal vesicles accumulate approx. 10 nmol of Ca2+/mg of protein and buffer ambient free Ca2+ at increasing concentrations (0.22-10.9 microM). Under these experimental conditions, microsomal glucose-6-phosphatase activity is unaffected by the concentration of extravesicular free Ca2+. 2. Different levels of intravesicular Ca2+ were obtained by treating microsomes with the Ca2+ ionophore A23187 and by stimulating active microsomal Ca2+ accumulation with Pi (3 mM). In both instances, microsomal glucose-6-phosphatase activity is unaffected by the level of intravesicular Ca2+.
Endothelin-1 mRNA expression in the rat kidney
Authors: Nunez D J R , Taylor E A , Oh V M S , Schofield J P , Brown M J
Published: August 2015

Cultured pig and bovine endothelial cells are capable of synthesizing endothelin-1 (ET-1). Thus the observation that the kidney contains a large number of binding sites for ET distributed in close proximity to endothelial cells suggests that ET-1 may be released from the endothelium to act locally on these receptors. In support of this hypothesis, using the technique of reverse transcription with specific amplification of cDNA, we report here that ET-1 mRNA is expressed in the rat kidney. The partial sequence of the amplified rat ET-1 cDNA confirms that the mature rat peptide is identical to that of the mouse, man and pig, but with some differences in codon usage.
Retinoic acid and transforming growth factor β differentially inhibit platelet-derived-growth-factor-induced Ito-cell activation
Authors: Davis B H , Rapp U R , Davidson N O
Published: August 2015

Sinusoidal Ito cells (stellate or fat-storing cells) undergo excessive cellular proliferation before the establishment and progression of hepatic fibrosis and cirrhosis. Retinoic acid and transforming growth factor beta (TGF beta) both inhibit Ito-cell [3H]thymidine incorporation in serum-containing media. Serum-induced mitogenicity was dependent on platelet-derived growth factor (PDGF). Additionally, pre-treatment of Ito cells with retinoic acid and TGF beta blocked PDGF-induced cell proliferation. TGF beta, but not retinoic acid, diminished PDGF-receptor and smooth-muscle alpha-actin abundance.
Dephosphorylation of autophosphorylated insulin and epidermal-growth-factor receptors by two major subtypes of protein-tyrosine-phosphatase from human placenta
Authors: Tappia P S , Sharma R P , Sale G J
Published: August 2015

The identity of protein-tyrosine-phosphatases (PTPases) active against autophosphorylated insulin receptor was probed by using an insulin-receptor-related peptide phosphorylated on tyrosine (peptide 1142-1153). Two major peaks of PTPase activity were resolved from the particulate (Triton X-100-soluble) fraction of human placenta by chromatography on DEAE-cellulose. The two peaks were purified 1300-2300-fold; other peaks of PTPase activity (greater than 15%) were not detected. Properties of the PTPases indicated that they corresponded to subtypes 1A and 1B. Both subtypes appeared capable of catalysing dephosphorylation of all autophosphorylation sites in three domains of the insulin receptor, with no appreciable difference in the pattern of dephosphorylation detected by two-dimensional tryptic-peptide mapping. The tyrosine-1150 domain of the insulin receptor in triply phosphorylated form was found to be highly sensitive to the action of both PTPases, and was dephosphorylated at least 4 times faster than the doubly and singly phosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains by either PTPase. This is significant, as the level of the triphosphotyrosine-1150 species has been shown to correlate well with the capacity of the insulin-receptor tyrosine kinase to phosphorylate other proteins. Both subtypes also dephosphorylated autophosphorylated epidermal-growth-factor (EGF) receptor by greater than 95%. Placental particulate (and cytosolic) PTPase activity against either receptor distributed approximately 2:1 between subtypes 1A and 1B as assayed in the presence of EDTA. In summary, PTPases within two major subtypes have been identified as phosphotyrosyl-insulin and -EGF-receptor phosphatases in vitro. The PTPases identified exhibit high affinities for substrates and high activities in cells, which is commensurate with the PTPases being important in vivo in controlling or reversing autophosphorylation-induced regulatory or signalling events.
Endotoxic lipid A induces intracellular Ca2+ increase in human platelets
Authors: Romano M , Molino M , Cerletti C
Published: August 2015

The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (tmax). Maximum [Ca2+]i levels, observed at 30 microM lipid A, were 432 +/- 60 nM. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of tmax, but shortening the time for 50% recovery (t50) from greater than 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1 microM], prolonging the tmax. to 54.7 +/- 5.1 s, but decreasing the t50 to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0.15 microM). These results suggest that platelet activation by lipid A involves an interaction between [Ca2+]i elevation and protein kinase C activation.
Guanine-nucleotide- and adenine-nucleotide-dependent regulation of phospholipase D in electropermeabilized HL-60 granulocytes
Authors: Xie M S , Dubyak G R
Published: August 2015

We have characterized the regulation of phospholipase D (PLD) in electropermeabilized HL-60 granulocytes in which endogenous phospholipids were pre-labelled with [3H]oleic acid. Treatment of these permeabilized cells with the non-hydrolysable GTP analogues guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta gamma-imido]triphosphate induced a sustained (near-linear for up to 60 min) accumulation of phosphatidic acid (PA). In the presence of ethanol a sustained production of phosphatidylethanol (PEt) was also observed. With increasing concentrations of ethanol, PEt formation increased, whereas PA formation declined; this indicated involvement of a PLD-type effector enzyme. The ability of GTP[S] to stimulate this PLD activity was Mg(2+)-dependent and was inhibited by GDP and its non-hydrolysable beta-thio analogue. Ca2+, at concentrations less than or equal to nM, had no effect on the GTP[S]-dependent PLD activity. However, higher concentrations of Ca2+ produced a significant potentiation of this activity. Inclusion of MgATP (greater than or equal to 0.1 mM), but not other nucleoside triphosphates, also induced a large potentiation of GTP[S]-dependent PLD activation. In the absence of guanine nucleotides, MgATP elicited no significant activation of PLD. Significantly, this effect of ATP was not mimicked by adenosine 5′-[beta gamma-methylene]triphosphate, a non-hydrolysable ATP analogue. Rather, this analogue inhibited both basal and ATP-potentiated GTP[S]-dependent PLD activity. This suggests that the ability of ATP to potentiate GTP[S]-dependent PLD activity involves phosphotransferase action rather than simple allosteric effects induced by adenine nucleotide binding. The absolute magnitude of the GTP[S]-dependent PLD activity which could be potentiated by MgATP was decreased by 90% when the permeabilized cells were preincubated for various times before addition of these stimulatory agents. This time-dependent loss of MgATP-induced potentiation was prevented when the permeabilized cells were preincubated in the presence of GTP[S]. These results demonstrate that electropermeabilized HL-60 granulocytes can be used to discriminate synergistic roles for a GTP-binding protein(s) and an ATP-dependent process (kinase?) in the regulation of phospholipase D activity.
5-Lipoxygenase is not essential in macrophage-mediated oxidation of low-density lipoprotein
Authors: Jessup W , Darley-Usmar V , O'Leary V , Bedwell S
Published: August 2015

The concentration-dependent effects of a series of lipoxygenase inhibitors and antioxidants on the macrophage-mediated oxidative modification of low-density lipoprotein (LDL) were measured. Their influence on macrophage 5-lipoxygenase pathway activity was also studied over the same concentration range. No correlation between inhibition of 5-lipoxygenase and of macrophage-mediated oxidation of LDL was observed. The capacity of the compounds to prevent cell-mediated modification of LDL could be explained in terms of their activity as either aqueous- or lipid-peroxyl radical scavengers. Two potent 5-lipoxygenase inhibitors (MK 886 and Revlon 5901), which had no radical-scavenging properties, were unable to block LDL modification. It is concluded that 5-lipoxygenase is not essential for LDL oxidation by macrophages.
Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction
Authors: Kitson T M
Published: August 2015

The effects of S-methyl diethyldithiocarbamate, S-methyl diethylmonothiocarbamate and bis(diethylcarbamoyl) disulphide on sheep liver cytoplasmic aldehyde dehydrogenase were investigated in vitro. The first compound has negligible effect. The second one is a weak inhibitor of the esterase activity of the enzyme and a weaker inhibitor of the dehydrogenase activity. A very low concentration of the third compound, however, acts as a potent inactivator of aldehyde dehydrogenase, similar in this respect to disulfiram, although somewhat slower to react. The possible involvement of these compounds in the physiological phenomenon known as the disulfiram ethanol reaction is discussed.
Citrate synthase from Mycobacterium smegmatis. Cloning, sequence determination and expression in Escherichia coli
Authors: David M , Lubinsky-Mink S , Ben-Zvi A , Suissa M , Ulitzur S , Kuhn J
Published: August 2015

A Mycobacterium smegmatis PstI library was constructed by cloning these fragments downstream from the lac promoter of the expression vector pHG171. Three identically sized clones were isolated by complementation of an Escherichia coli strain (chi 2338) deficient in citrate synthase. One insert (pBL265) was used in hybridization experiments with DNA from E. coli and M. smegmatis and it was demonstrated that the clones were indeed from M. smegmatis. The transcription of the M. smegmatis citrate synthase gene in E. coli relied upon the lac promoter. In translation experiments performed in vitro pBL265 gave rise to a novel protein of about 42 kDa. This band was not seen in ‘opposite-orientation’ subclones. Various subclones in which the 5′-end was shortened nevertheless complement E. coli chi 2338 and produce the 42 kDa protein. This demonstrates that the M. smegmatis citrate synthase gene uses its own ribosome-binding site in E. coli. The relevant 1.8 kb of the 2.8 kb insert was sequenced. A consensus E. coli ribosome-binding site was found centred precisely 10 bp upstream of the methionine codon. Other interesting features revealed by the sequence are discussed. Citrate synthase activity was assayed in vitro and the mycobacterial enzyme was found to be similar to those of the Gram-positive bacteria.
Phospholipase A2 in human ascitic fluid. Purification, characterization and immunochemical detection
Authors: Kortesuo P T , Nevalainen T J
Published: August 2015

A phospholipase A2 (PLA2, EC 3.1.1.4) was purified from human cell-free ascitic fluid (a-PLA2) by ion-exchange chromatography and h.p.l.c. on a reverse-phase column to apparent homogeneity. The enzyme had an Mr of approx. 10,000 as determined by SDS/PAGE. Polyclonal antibodies raised in a rabbit were specific to a-PLA2, as judged by immunoblotting. A time-resolved fluoroimmunoassay (TR-FIA) for measuring the concentration of a-PLA2 in various body fluids was developed. The detection limit of the assay was about 6 ng/ml. The antiserum did not cross-react with pancreatic secretory phospholipase A2 as measured by TR-FIA. The enzyme content was studied in various samples, including normal human serum, buffy-coat leucocytes, synovial fluid, and pancreas and spleen homogenates.
Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3
Authors: Hsieh J C , Huang S C , Chen W L , Lai Y C , Tam M F
Published: August 2015

Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.
Algorithms for the derivation of Flux and Concentration Control Coefficients
Authors: Schulz A R
Published: August 2015

A computer program is described that derives the Flux and Concentration Control Coefficients for linear and branched metabolic pathways. The program prompts the user to enter a concise description of the metabolic pathway. From this description, the program constructs the appropriate equations in matrix form. The algorithm employed to obtain the symbolic determinants is described, and this algorithm also provides a convenient method for manual derivation of the Control Coefficients. The computer-based method will accommodate unlimited feed-forward and feed-backward loops and a maximum of two branches from each metabolite on the main pathway. The utility of the method is illustrated with a linear path with feed-forward and feed-backward loops, and with a substrate cycle as an example of a path with two branches.
Quantification of glucose cycling and the extent of equilibration of glucose 6-phosphate with fructose 6-phosphate in islets from ob/ob mice
Authors: Chandramouli V , Khan A , Ostenson C G , Berggren P O , Löw H , Landau B R , Efendić S E
Published: August 2015

Pancreatic islets from fed and fasted obese hyperglycaemic (ob/ob) mice were incubated with [1-14C]glucose at 5.5 mM and 16.7 mM, [1-14C]mannose at 16.7 mM, and 3H2O. Yields of 14CO2 and 14C-labelled lactate, and amounts of 14C from [1-14C]mannose incorporated into glucose and of 3H bound to C-2 of glucose, were measured. Glucose utilization was determined from yields of 3H2O from [5-3H]glucose. From the results using 14C, 32-43% of the hexoses phosphorylated to hexose 6-phosphate were estimated to have been dephosphorylated, i.e. cycled. Estimates of hexose cycling from 3H incorporation into glucose were similar. Differences in the ratios of 14C yields in CO2 and lactate indicated incomplete isotopic equilibration between glucose 6-phosphate and fructose 6-phosphate, making the estimates of cycling semi-quantitative. In the fasted state, a larger percentage of the hexose utilized went to lactate than in the fed state. Thus conversion of mannose into glucose in islets indicates the occurrence of glucose cycling in islets. Yields of 14C from [1-14C]mannose, compared with from [1-14C]glucose, provide an approach for quantifying the extent of this cycling. These data provide further evidence for extensive glucose cycling occurring in ob/ob islets in both the fed and the fasted state.
Synthesis of a gene for rat liver fatty-acid-binding protein and its expression in Escherichia coli
Authors: Worrall A F , Evans C , Wilton D C
Published: August 2015

A gene coding for rat liver fatty-acid-binding protein (FABP) has been constructed by the single-step ligation of ten synthetic oligonucleotides. The gene has been cloned into the bacterial expression vector pKK223-3. Induction of protein synthesis from this gene results in over expression of an FABP that is indistinguishable in its structure and binding properties with that isolated from rat liver.
Phorbol-ester-mediated expression of the collagen type I pro-α2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant
Authors: Stuiver I , Shimizu Y , Shimizu N
Published: August 2015

We have previously isolated a phorbol 12-myristate 13-acetate (PMA)-non-responsive variant line (VT-1) from mouse 3T3-L1 cells [Shimizu, Fujiki, Sugimura & Shimizu (1986) Cancer Res. 46, 4027-4031]. Differential hybridization of cDNAs obtained from PMA-treated 3T3-L1 and VT-1 cells resulted in the isolation of a number of unique cDNA clones, including one with a high degree of sequence similarity to the type I pro-alpha 2 collagen gene (COL1A2) [Amagai, Inokuchi, Nishikawa, Shimizu & Shimizu (1989) Somat. Cell Mol. Genet. 15, 153-158]. Here we examined the expression of type I collagen pro-alpha 2 [pro-alpha 2(I)] mRNA and production of type I collagen in these two cell lines. In quiescent cells, the pro-alpha 2(I) steady-state mRNA levels were four times greater in 3T3-L1 cells than in VT-1 cells. PMA addition caused the steady-state levels of pro-alpha 2(I) mRNA to be six times greater in 3T3-L1 cells than in VT-1 cells, with a maximum at 30-60 min. The pro-alpha 2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by PMA treatment, but no significant increase was detected in VT-1 cells. The correlation of collagen expression with a PMA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction.
Inositol 1,3,4,5-tetrakisphosphate-induced Ca2+ sequestration into bovine adrenal-medullary secretory vesicles
Authors: Yoo S H
Published: August 2015

Ins(1,3,4,5)P4 induced a rapid sequestration of Ca2+ into both secretory vesicles and microsomes of bovine adrenal medulla. The Ca(2+)-sequestering role of Ins(1,3,4,5)P4 contrasts with the Ca(2+)-releasing role of Ins(1,4,5)P3 in adrenal-medullary secretory vesicles and microsomes. The Ins(1,3,4,5)P4-induced Ca2+ sequestration into secretory vesicles was not inhibited by heparin (50 micrograms/ml), whereas Ins(1,4,5)P3-induced Ca2+ release was completely inhibited, indicating two different receptors for Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Furthermore, Ins(1,3,4,5)P4 was as effective at 4 degrees C as at 24 degrees C in sequestering Ca2+ into secretory vesicles, implying Ca2+ sequestration through receptor-operated Ca2+ channels or activation of the Ca(2+)-exchange mechanism by Ins(1,3,4,5)P4. The Ca(2+)-sequestering activity of Ins(1,3,4,5)P4 has also been demonstrated with 45Ca2+; 10 microM-Ins(1,3,4,5)P4 induced rapid uptake of 45Ca2+ into secretory vesicles optimized for Ca2+ uptake, whereas 10 microM-Ins(1,4,5)P3 induced 45Ca2+ release from secretory vesicles in similar experiments.