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Biomedical subjects

R K Sharma

Publications and source records attributed to R K Sharma.

At least 307 records · Page 17Linked to original sources
PubMed1993

Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Adenosine Triphosphate↗
PubMed1993

Purification and characterization of bovine brain calmodulin-dependent protein kinase. II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme.

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca2+ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca2+. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca2+ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca2+ flux via the protein kinase autophosphorylation mechanism.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Ginsenosides are potent and selective inhibitors of some calmodulin-dependent phosphodiesterase isozymes.

The effects of various ginsenosides on calmodulin-dependent phosphodiesterase isozymes have been investigated. Ginsenosides were found to be potent inhibitors of bovine heart calmodulin-dependent phosphodiesterase and the 60-kDa isozyme of bovine brain calmodulin-dependent phosphodiesterase but not of the 63-kDa isozyme of bovine brain calmodulin-dependent phosphodiesterase. Since the inhibition of phosphodiesterase by ginsenosides was overcome by increasing the concentration of calmodulin, this suggests that ginsenosides act specifically and reversibly against the action of the calmodulin. These compounds therefore should be valuable tools to investigate the diverse physiological roles of distinct phosphodiesterase isozymes.

Animals↗
PubMed1993

Identification, purification and characterization of a membrane-associated N-myristoyltransferase inhibitor protein from bovine brain.

N-Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme that catalyses the covalent transfer of myristic acid from myristoyl-CoA to the N-terminal glycine residue of a protein substrate. Subcellular fractionation of bovine brain indicates that NMT activity was located in both the cytosolic and the particulate fraction of the cell. Removal of the particulate fraction resulted in a 2-fold enhancement of NMT activity. Reconstitution of the particulate fraction and cytosolic fraction resulted in inhibition of the elevated cytosolic NMT activity. These results indicated the existence of putative inhibitor(s) activity of NMT located in the particulate fraction of bovine brain. The inhibitor was stable to heat and was identified as a protein, on the basis of its susceptibility to the proteases trypsin and chymotrypsin. Protease degradation first required the delipidation of the particulate fraction. The inhibitor was purified to near-homogeneity by heat treatment, solvent extraction and Sephacryl S-300 gelfiltration column chromatography. The inhibitor was purified 630-fold from the particulate fraction with a 20% yield. The protein inhibitor had an apparent molecular mass of 92 kDa by gel filtration and 71 kDa by SDS/PAGE, indicating the protein is monomeric. The inhibitor did not interact directly with myristoyl-CoA and possessed no protease, thioesterase or demyristoylase activity. Purified inhibitor protein inhibited the formation of 1167 pmol of myristoyl-peptide/min per mg of protein.

Acyl Coenzyme A↗
PubMed1993

Bovine brain calmodulin-dependent protein kinase II: molecular mechanisms of autophosphorylation.

A bovine brain calmodulin-dependent protein kinase undergoes autophosphorylation with a maximal incorporation of 10 mol phosphate per mol enzyme. The autophosphorylated enzyme is fully active, independent of Ca2+ and calmodulin. Although the autophosphorylation reaction depends on Ca2+ and calmodulin, it can be rendered Ca(2+)-independent after about 15 seconds when 1 mol phosphate has been incorporated per mol enzyme. This Ca(2+)-independent autophosphorylation is an intramolecular reaction, since mixing of autophosphorylated and non-phosphorylated calmodulin-dependent protein kinase does not result in the Ca(2+)-independent phosphorylation of the non-phosphorylated enzyme. The Ca(2+)-independent autophosphorylation can be inhibited by an exogenous substrate, e.g., casein. The casein phosphorylation activity is approximately proportional to the level of phosphorylation of the enzyme.

Animals↗
PubMed1993

Interaction of atrial natriuretic factor and endothelin-1 signals through receptor guanylate cyclase in pulmonary artery endothelial cells.

The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [125I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC50 of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC50 of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.

Animals↗
PubMed1993

Cloning and expression of an ATP-regulated human retina C-type natriuretic factor receptor guanylate cyclase.

The natriuretic factors are structurally related polypeptide hormones that regulate the hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. Presumably, these hormones act through the activation of guanylate cyclases which are also the specific receptors of these hormones. Two such structurally similar cell surface receptors are known; the ligand for one is atrial natriuretic factor (ANF) and for the other is C-type natriuretic peptide (CNP). Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that its ligand binding site is extracellular and the catalytic site is intracellular, but the mere ligand binding to the receptor domain does not activate the cytosolic catalytic domain. An intervening ATP-mediated event is obligatory: ATP binds to a defined ATP-regulated module (ARM) sequence and bridges the events of ligand binding and signal transduction. The mechanism of CNP signaling is not known, although CNP in intact cells transfected with CNP receptor guanylate cyclase (CNP-RGC) stimulates the formation of cyclic GMP. Furthermore, there is no prior evidence of the presence of CNP signal transduction system in retina, although the presence of ANF-RGC has been documented. We now report the molecular cloning and expression of CNP-RGC from human retina and show that ATP is obligatory in CNP signaling also.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Elevated N-myristoyl transferase activity is reversed by sodium orthovanadate in streptozotocin-induced diabetic rat.

N-Myristoyl transferase (NMT) is the enzyme that covalently modifies several proteins important in signal transduction. Streptozotocin-induced diabetes resulted in a 2-fold increase in NMT activity from rat liver as compared to control animals. Administration of sodium orthovanadate to the diabetic rats reduced the activity of the NMT to 75-120% of the control values. Elevated NMT activity was observed with both cAMP-dependent protein kinase-derived and pp60src-derived peptide substrates. No significant change in the apparent Km was observed with the cAMP-dependent protein kinase-derived peptide substrate. Unlike in rat brain, in all conditions highest NMT activity was observed in the particulate fraction of rat liver.

Acyltransferases↗
PubMed1993

Core sequence of ATP regulatory module in receptor guanylate cyclases.

Atrial natriuretic factor (ANF) and C-type natriuretic peptide (CNP)-activated guanylate cyclases are single-chain transmembrane-spanning proteins, containing both ligand binding and catalytic activities. In both proteins, ligand binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that obligatory in this activation process is an ATP-dependent step. ATP directly binds to the cyclase and bridges the events of ligand binding and signal transduction. A defined ATP-regulated module (ARM) sequence (Gly503-Arg-Gly-Ser-Asn-Tyr-Gly509) in the cyclase is critical in the ATP-mediated event. Through genetic remodeling techniques, we have now identified the core ARM sequence that is essential in both ANF and CNP signaling. This sequence is Gly-Xa-Xa-Xa-Gly, represented by Gly505-Ser-Asn-Tyr-Gly509 in the case of ANF-RGC ARM and by Gly499-Ser-Ser-Tyr-Gly503 in the CNP receptor guanylate cyclase ARM.

Adenosine Triphosphate↗
PubMed1993

Effect of orotic acid on beta 1,4-galactosyltransferase during liver regeneration.

The study was designed to understand the effect of orotic acid (OA) on the expression of beta 1,4-galactosyltransferase (GalTase), an enzyme involved in the transfer of galactose from UDP-galactose to a non-reducing terminal N-acetylglucosamine of a glycoprotein or glycolipid. Rats were fed a semisynthetic diet containing 1% OA for 2 weeks and the livers were stimulated to regenerate by two-thirds partial hepatectomy (PH). The level of activity of the enzyme and the steady-state level of hepatic mRNA transcripts of GalTase were determined prior to PH and at 12, 24, 48, 72 h and 10 days after the surgery. The data show that the hepatic activity of GalTase is unaltered in both the control and OA-fed groups until 12 h following surgery, but begins to increase after this time period. In the control group a progressive increase was seen throughout the experimental period following PH. On the other hand in the OA-fed group 24 h after PH the initial increase seen up to 24 h was arrested later on and the activity remained inhibited throughout the rest of the experimental period. The supplementation of 1% OA diet with 0.3% adenine, which is known to reverse the OA-induced imbalance in the nucleotide pool sizes, relieved the inhibition of GalTase activity. The steady-state level of hepatic mRNA paralleled the activity of GalTase at all the time points studied during liver regeneration. The reduction in the level of mRNA transcripts of GalTase in the OA-fed group may not be due to either a general inhibition of synthesis and/or degradation of mRNAs as revealed by a comparison of the expression of beta-galactoside 2,6-sialyltransferase in both the control and OA-fed groups. The study thus suggests an imbalance in nucleotide pools, such as the one induced by OA, may play a role in the regulation of glycosylation by modulating the glycosyltransferases.

Adenine↗
PubMed1993

Prolonged peritoneal dialysis in acute renal failure using Tenckhoff catheter.

The changing spectrum of acute renal failure (ARF), in children has necessitated the support by peritoneal dialysis (PD) for a longer duration. Use of permanent catheters like Tenckhoff catheter can overcome many of the problems associated with the prolonged use of temporary catheters. Thirty one infants and children with mean (SD) age of 49.3(39.4) months were subjected to PD using surgically placed Tenckhoff catheter. Catheters were put under general anesthesia in 28 patients and under local anesthesia in 3 patients. The technique of catheter insertion has been described in detail. All the catheters, except one, functioned immediately after the insertion. There was no intraperitoneal hemorrhage, dialysate leak or poor catheter drainage. The mean (SD) duration of catheter placement for PD was 11.3 (16.1) days (range 2.90 days). There were 6 episodes of infections (19.2%), peritonitis in 4 (12.8%) and exit site infection in 2 (6.4%). In 2 patients, infection episodes did not respond to antibiotics and the catheter had to be removed. There was no mortality due to complications of PD procedure and catheter insertion. Acute intermittent PD using surgically placed Tenckoff catheter was done safely for prolonged duration and also had additional advantages of good catheter drainage and no intraperitoneal hemorrhage.

Acute Kidney Injury↗
PubMed1993

Acute renal failure in the elderly: experience from a single centre in India.

A total of 139 patients with acute renal failure (ARF) were studied, of which 41 (29.4%) were elderly with mean age of 67.1 years and 98 (70.6%) were younger with mean age of 32.3 years. Surgical causes accounted for 65% of geriatric ARF while medical causes were predominant in the younger patients (55.1%). Amongst the surgical causes, prostate-related problems due to obstruction or following transurethral resection of prostate were seen in 20 patients (74%). Drugs and sepsis were the predominant causes of medical ARF in the geriatric patients (85.7%). Of all the causes of geriatric ARF, which included both medical and surgical, nephrotoxic drugs either alone or in combination with other predisposing factors were the cause in 22 (51%) patients. Haemodialysis was needed in 15 of geriatric (36.6%) and 64 of younger (65.3%) ARF patients. Recovery from ARF, as evidenced by normalization of serum creatinine, was delayed in the elderly as compared to the younger patients (32.0 versus 11.4 days, P < 0.001). Mortality, though higher in the elderly as compared to the younger patients, was not significantly different (9.75% versus 6.1%).

Acute Kidney Injury↗
PubMed1992

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Animals↗
PubMed1992

Demonstration of multiple forms of bovine brain myristoyl CoA:protein N-myristoyl transferase.

Four distinct N-myristoyl transferase (NMT) activity peaks, designated I, II, III, and IV, were separated from the cytosolic fraction of bovine brain by DEAE-Sepharose column chromatography. Peaks I, II, III and IV were characterised biochemically with respect to substrate specificity: with cAMP-dependent protein kinase and pp60src derived peptides, and by their apparent molecular mass. The apparent molecular mass of peaks I, II, III and IV were 190 kDa, 224 kDa, 390 kDa and 76 kDa, respectively. These results indicate that bovine brain contains multiple forms of NMT.

Acyltransferases↗
PubMed1992

Nitroprusside-sensitive and insensitive guanylate cyclases in retinal rod outer segments.

Rod outer segments of retina contain guanylate cyclase activity both in the cytosol and membrane fractions. Though the activity in the cytosol is a small fraction of the total activity, it is highly activated by nitroprusside, a nitric oxide generating agent. The membrane guanylate cyclase on the other hand is unaffected by nitroprusside both before and after solubilization. The effects of nitroprusside or nitric oxide on photoreceptor function should therefore be mediated by the cytosolic and not the membrane guanylate cyclase.

Animals↗
PubMed1992

A structural motif that defines the ATP-regulatory module of guanylate cyclase in atrial natriuretic factor signalling.

Atrial natriuretic factor (ANF)-dependent guanylate cyclase is a single-chain transmembrane-spanning protein, containing an ANF receptor and having catalytic activity. ANF binding to the receptor domain activates the catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening step regulated by ATP, but its mechanism is not known. Through a programme of site-directed and deletion mutagenesis/expression studies, we report herein the identity of a structural motif (Gly503-Arg-Gly-Ser-Asn-Tyr-Gly509) that binds ATP and amplifies the ANF-dependent cyclase activity; this, therefore, represents an ATP-regulatory module (ARM) of the enzyme, which plays a pivotal role in ANF signalling.

Adenosine Triphosphate↗