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K Murakami

Publications and source records attributed to K Murakami.

At least 1,063 records · Page 59Linked to original sources
PubMed1986

Mechanism of inhibitory action of sodium nitroprusside in vascular smooth muscle of rabbit aorta.

Mechanism of inhibitory action of sodium nitroprusside in vascular smooth muscle of rabbit aorta was examined. Sodium nitroprusside inhibited the 1 X 10(-6) M norepinephrine-induced contraction with IC50 (concentration to induce 50% inhibition) of 1.40 X 10(-7) M, but had little effect on the 65.4 mM K-induced contraction. In contrast, verapamil had little effect on the norepinephrine-induced contraction at the concentration needed to inhibit the high K-induced contraction. The inhibitory effect of sodium nitroprusside on the norepinephrine-induced contraction did not change in the verapamil-treated aorta. In the high K-solution with verapamil, norepinephrine induced a sustained contraction with similar magnitude as that in normal solution. Sodium nitroprusside also inhibited this contraction although the inhibitory effect was slightly less compared to that in the polarized aorta (IC50 = 2.90 X 10(-7) M, p less than 0.01). In a Ca-deficient solution, norepinephrine induced a transient contraction due to release of cellular Ca. Sodium nitroprusside inhibited the norepinephrine-induced transient contraction at slightly higher concentrations than those which inhibit the sustained contraction (IC50 = 5.55 X 10(-7) M). In a high K, Ca deficient solution, norepinephrine also induced a transient contraction. The inhibitory effect of sodium nitroprusside on the transient contraction was reduced in the depolarized aorta (IC50 = 9.50 X 10(-6) M). It is suggested that the inhibition of norepinephrine-induced sustained contraction by sodium nitroprusside is mainly due to the direct inhibitory effect on the receptor linked-Ca channels whereas the inhibition of norepinephrine-induced transient contraction is attributable mainly to membrane hyperpolarization and, at higher concentrations, to a direct inhibition of Ca release.

Animals↗
PubMed1986

Phosphoprotein F1: purification and characterization of a brain kinase C substrate related to plasticity.

To study the role of protein kinase C (PKC) and its substrates in neuronal function, we have investigated the in vitro endogenous phosphorylation of the neuronal phosphoprotein F1 after induction of synaptic plasticity by long-term potentiation (LTP). The protein F1 phosphorylation was found to increase 5 min (Routtenberg et al., 1985), 1 hr (Lovinger et al., 1986) and 3 d (Lovinger et al., 1985) after LTP. The characteristics of this protein bear close similarities to a number of proteins characterized in various neuronal systems, such as B50 (brain specific, synaptosome-enriched protein), pp46 (a growth cone protein), and GAP 43 (nerve growth and regeneration-associated protein). A positive identification of the purified protein F1 with these proteins would link protein F1 to the developmental growth of axons, nerve regeneration, and polyphosphoinositide metabolism, as well as adult plasticity. We have therefore purified and partially characterized native protein F1 so that a meaningful comparison among the properties of these proteins can be made. Using synaptosomal plasma membrane (P2') as starting material, subsequent purification involved pH extraction, 40-80% ammonium sulfate precipitation, hydroxylapatite, and phenyl-Sepharose column chromatography. This procedure achieved greater than 800-fold purification and about 45% yield relative to P2'. Purified protein F1 (Mr = 47,000, pI = 4.5) was found to be a hydrophilic molecule and was phosphorylated by 1000-fold purified PKC in the presence of phosphatidylserine (PS) and Ca2+. The Ka of PS activation is about 15 micrograms/ml (approximately 20 microM), and that of Ca2+ is about 25 microM. Diolein and DiC:8 (a synthetic diacylglycerol) lowered the requirement of Ca2+ for maximal stimulation from 100 to 5 microM. Ca2+-calmodulin kinases type I and II did not phosphorylate protein F1. The phosphoamino acid analysis showed that 97% of the total incorporated 32P-phosphate was on the serine residue. Phosphopeptide mapping using V8-protease generated 2 phospho-fragments having apparent Mr of 13,000 and 11,000. Calmodulin at 3.6 microM inhibited 95% of protein F1 phosphorylation by PKC. The availability of purified native protein F1 should facilitate investigation of the physiological role of this protein in the nervous system and its functional regulation by PKC.

Animals↗
PubMed1985

In vitro biosynthesis of human renin and identification of plasma inactive renin as an activation intermediate.

The biosynthesis and post-translational modifications, including proteolytic processing and core glycosylation, of the human renin precursor have been studied in vitro in a cell-free system. For this purpose, highly enriched renin mRNA was isolated from a renin-producing juxtaglomerular cell tumor and translated in rabbit reticulocyte lysate containing [35S]methionine in the presence or absence of dog pancreas microsomal membranes. Fluorographic analysis of the radioactive translation products, immunoprecipitated and then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the primary translation product, preprorenin (Mr = 45,000), is initially processed to glycosylated prorenin (Mr = 47,000) during or shortly after its sequestration into the lumen of the microsomal membranes. The vectorial translocation across the membrane was confirmed by the observation that the proform was resistant to digestion with trypsin while preprorenin was sensitive. Radiosequencing and the use of prorenin-specific antibodies established the cleavage points of the pre- and profragment and showed that the in vitro precursor of human renin contains a 23-residue signal peptide and a 43-residue prosegment. The post-translational modification which, despite the removal of signal peptide, resulted in an increase in apparent Mr, reflects the glycosylation as examined using Xenopus oocytes microinjected with renin mRNA in the presence of tunicamycin, an inhibitor of protein glycosylation. Four anti-peptide antibodies which specifically recognize the NH2 terminus (Pro 1), two middle parts (Pro 2A and Pro 2B), and COOH terminus (Pro 3) of the prosegment, respectively, have been raised and used to characterize plasma prorenin. Renin precursors (pre- and prorenin) synthesized in vitro or in the kidney reacted with these antibodies (anti-Pro 1, anti-Pro 2A, anti-Pro 2B, and anti-Pro 3). However, quite unexpectedly, human plasma prorenin was recognized only by anti-Pro 3, indicating that plasma prorenin is a truncated version of intact prorenin, which lacks a large portion of the NH2 terminus of the prosegment and may represent an activation intermediate. This somewhat surprising result may lead to a better understanding of the exact roles and activation mechanisms of plasma prorenin existing in a relatively large amount.

Amino Acid Sequence↗
PubMed1985

Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2+.

Unsaturated fatty acids (oleic acid and arachidonic acid) activate purified protein kinase C independently of phospholipid and Ca2+. Oleic acid activation of protein kinase C is as effective as phosphatidylserine and Ca2+. Ka values for oleic acid and arachidonic acid are 50 and 53 microM, respectively. In contrast to the cis fatty acids, a trans form (elaidic acid) or a saturated fatty acid (stearic acid) has little or no effect on protein kinase C activation. If cis fatty acid liberation is physiologically important, this suggests that another mechanism may exist for protein kinase C activation, in addition to phospholipase C/phosphatidylinositol turnover signaling, possibly via the liberation of cis fatty acids by the Ca2+-dependent phospholipase A2 system.

Animals↗
PubMed1985

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex.

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.

Adrenal Cortex↗
PubMed1985

Stabilization of the adenylate energy charge by the depletion of adenylates without glycolytic stimulation.

A relationship between the AMP deaminase reaction and the recovery of the energy charge was examined using permeabilized yeast cells. Citrate inhibited the glycolytic flux and the recovery of the energy charge. The addition of spermine enhanced the recovery of the energy charge without the reversal of the inhibition of glycolysis in the presence of excess citrate. The AMP deaminase reaction can participate in the stabilization of the energy charge only by the depletion of total adenylates, and not by the glycolytic stimulation under the conditions where citrate is highly increased during aerobic growth conditions.

AMP Deaminase↗
PubMed1985

Role of AMP deaminase reaction in the control of fructose 1,6-bisphosphatase activity in yeast.

The physiological role of the inhibition of AMP deaminase (EC 3.5.4.6) by Pi was analyzed using permeabilized yeast cells. (a) Fructose 1,6-bisphosphatase (EC 3.1.3.11) was inhibited only a little by AMP, which was readily degraded by AMP deaminase under the in situ conditions. (b) The addition of Pi, which showed no direct effect on fructose 1,6-bisphosphatase, effectively enhanced the inhibition of the enzyme by AMP increased through the inhibition of AMP deaminase. (c) Pi activated phosphofructokinase (EC 2.7.1.11) and inhibited AMP deaminase activity. AMP deaminase reaction can act as a control system of fructose 1,6-bisphosphatase activity and gluconeogenesis/glycolysis reaction through the change in the AMP level. Pi may contribute to the stimulation of glycolysis through the inhibition of fructose 1,6-bisphosphatase by the increase in AMP in addition to the direct activation of phosphofructokinase.

AMP Deaminase↗
PubMed1985

AMP deaminase reaction as a control system of glycolysis in yeast. Role of ammonium ion in the interaction of phosphofructokinase and pyruvate kinase activity with the adenylate energy charge.

The role of ammonium ion and AMP deaminase (EC 3.5.4.6) reaction in the activation of phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) by the decrease in the adenylate energy charge was investigated using permeabilized yeast cells. Response of AMP deaminase, phosphofructokinase, and pyruvate kinase to variation in the energy charge is typical of the ATP-regenerating enzymes: an activation with the decrease in the energy charge under the in situ conditions. The addition of polyamine activated AMP deaminase in situ, resulting in the subsequent increase in ammonium production, which can stimulate the phosphofructokinase activity with the increase in the optimal energy charge value giving maximal activity of the enzyme. The optimal energy charge value of phosphofructokinase was 0.2-0.25 in the absence of ammonium ion and was shifted to the value above 0.5 by the addition of ammonium ion, whereas Pi, an activator of the enzyme showed little effect on the increase in the optimal energy charge value. The optimal energy charge value of AMP deaminase and pyruvate kinase was not affected by the addition of their effectors. Modulation of the response to the energy charge of phosphofructokinase and pyruvate kinase was analyzed in terms of the "activation coefficient," which was defined as the ratio of the activity at the energy charge of 0.6 to that at the value of 0.9. Activation of phosphofructokinase by the physiological decrease in the energy charge (0.9 to 0.6) can be enhanced by the increase in ammonium ion specifically, although the coefficient of pyruvate kinase remained unaffected by ammonium ion. These results suggest that the AMP deaminase reaction as an ammonium-forming reaction can participate in a key role in the stimulation of phosphofructokinase or glycolytic flux in cells.

AMP Deaminase↗
PubMed1985

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats.

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.

Animals↗
PubMed1985

Probing the topography of lectins with site-specific spin-labeled glycosides.

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.

Electron Spin Resonance Spectroscopy↗
PubMed1985

Monoclonal antibodies against species-specific cephalosporinase of Pseudomonas aeruginosa.

Ten hybridomas which produced monoclonal antibodies against species-specific cephalosporinase of Pseudomonas aeruginosa were constructed by somatic cell fusion of SP-2/0 myeloma cells with spleen cells from hyperimmunized BALB/c mice and intraperitoneally injected into mice. Monoclonal antibodies were partially purified from ascites fluids. All ten antibodies thus prepared were IgG immunoglobulins. In a solid-phase immunoassay the antibodies gave positive reactions which could be prevented by the presence of free cephalosporinase. When the antibodies were precipitated with anti-(mouse IgG), the cephalosporinase activity was co-precipitated. Also, these antibodies were co-eluted with cephalosporinase activity in gel filtration. Nine of the monoclonal antibody preparations elevated the cephalosporinase activity by about 6-40% and only one inhibited it by about 50%. All the monoclonal antibodies were highly specific to P. aeruginosa cephalosporinase and showed no cross-reaction with nine cephalosporinases and four penicillinases of other gram-negative bacteria.

Antibodies, Monoclonal↗
PubMed1985

Identification of plasma inactive renin as prorenin with a site-directed antibody.

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.

Antibody Formation↗
PubMed1985

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin.

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.

Azo Compounds↗