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R L Khandelwal

Publications and source records attributed to R L Khandelwal.

At least 73 records · Page 4Linked to original sources
PubMed1982

Glycogen metabolizing enzymes in brain.

Three enzymes, glycogen phosphorylase, glycogen synthase, and phosphoglucomutase were evaluated in subcellular fractions and in brain regions. Also the development of each of these enzymes was evaluated in whole brain homogenates. Each enzyme increased during the first three weeks of post partum in a manner that is similar to the development of glycolytic enzymes during this period. The specific activity of each enzyme in various subcellular fractions indicated that the enzymes were primarily soluble. Also unlike the glycolytic enzyme phosphoglycerate kinase, the glycogen metabolizing enzymes had a lower specific activity in synaptosomes than in particle free supernatant fractions of homogenates. Regarding regional distribution small (less than twofold) but significant differences were seen between different brain areas. An inverse relationship between the glycogen metabolizing enzymes and hexokinase was observed, that is, regions highest in glycogen synthase and glycogen phosphorylase were lowest in hexokinase and regions highest in hexokinase were lowest in the glycogen metabolizing enzymes.

Age Factors↗
PubMed1982

Glycogen metabolizing enzyme activities in the developing rat liver.

1. The glycogen content of rat liver increased prenatally and dramatically decreased after the birth. 2. Glycogen synthase and glycogen phosphorylase activities increased prenatally, declined at 12 hr after birth and then again increased. 3. Phosphorylase kinase activities did not significantly change prenatally but steadily increased after the birth. 4. Protein kinase activities (units/mg liver) did not change prenatally but slightly increased after the birth. 5. Phosphoprotein phosphatase activities using phosphorylase a and histone as substrates dramatically increased at birth and then decreased after 24 hr of the birth.

Aging↗
PubMed1982

Inorganic pyrophosphatase activity in oral streptococci: purification and properties of the enzyme from Streptococcus salivarius.

Inorganic pyrophosphatase has been purified from the soluble fraction of Streptococcus salivarius by protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified approximately 500-fold with a 33% yield. The purified enzyme was homogeneous since it showed a single band when examined by nondenaturing polyacrylamide gel electrophoresis. It was rich in acidic (glutamic and aspartic) amino acids, as well as serine and glycine. The enzyme was devoid of sulfur-containing amino acids. The purified enzyme was specific for the hydrolysis of inorganic pyrophosphate and did not hydrolyze any other phosphate-ester compound examined. Inorganic pyrophosphatase activity was completely dependent on a divalent cation. Activity was maximum in the presence of Mg2+ while activity in the presence of Mn2+ and Co2+ was significantly lower. In the presence of Mg2+, a number of divalent cations, however, inhibited the enzyme activity. The true substrates for S. salivarius inorganic pyrophosphatase were magnesium-pyrophosphate complexes, i.e., MgPPi and Mg2PPi, while free Mg2+ had no effect on the enzyme activity and free PPi inhibited the hydrolysis of inorganic pyrophosphate. Km value for magnesium-pyrophosphate complexes was 16.4 microM. Km value for total Mg2+ was similar ranging between 14.4 and 20 microM. Analysis of data by Hill plots indicated one binding site for Mg2+ and two for PPi. Among various nucleotides and glycolytic intermediates examined, GDP, GMP, and fructose-1, 6-P2 showed significant inhibitory effect on enzyme activity.

Amino Acids↗
PubMed1980

Dissociation of rat liver high molecular weight phosphorylase phosphatase by salt.

The chromatography of rat liver crude extract (10 000 x g supernatant) on a Biogel A-0.5 m column resulted in two peaks of phosphorylase phosphatase activity. The first peak, eluted in void volume, is of microsomal origin whereas the second peak, a cytoplasmic origin, was eluted with an apparent molecular weight of 260 000. In the presence of NaCl, the cytosolic enzyme was dissociated into intermediary (Mr = 135 000 and 68 000) and low molecular weight (Mr = 35 000) forms with an apparent activation of the enzyme activity. Similar dissociation was observed in the presence of KCl and (NH4)2SO4. A partially purified high molecular weight phosphorylase phosphatase was also dissociated into the low molecular weight form by 0.2 M NaCl. However, a protein phosphatase showing activity towards 32P-labelled histone was not dissociated by 0.2 M NaCl or 0.1 M (NH4)2SO4.

Animals↗
PubMed1980

Studies on in activation and reactivation of homogeneous rabbit liver phosphoprotein phosphatases by inorganic pyorphosphate and divalent cations.

Preincubation of two homogeneous rabbit liver phosphoprotein phosphatases (phosphophoprotein phosphohydrolases, EC 3.1.3.16) (Khandelwal, R.L., Vandenheede, J.R. and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) with ATP, ADP and PPi caused a time- and concentration-dependent inactivation of the enzyme activity. A 50% inactivation of phosphoprotein phosphatase I required relatively low concentration of inactivating metabolite and less preincubation time as compared to the inactivation of phosphoprotein phosphatase II. AMP, adenosine, adenine, Pi, EDTA, EGTA, 1,10-phenanthroline and diethyl dithiocarbamate were without effect on both enzymes. Pretreatment of both enzymes by metal-chelating agents followed by PPi did not augment the effect observed with PPi alone. Both inactivated enzymes could be reactivated by cobalt or manganese in the presence of dithiothreitol. Although the extent of reactivation by these two metal ions was almost similar, cobalt required a ten times lower concentration than manganese for this process. No difference in inactivation or reactivation of both enzymes was observed with different substrates, phosphorylase a, histone or casein, employed in the assay. Pi and PPi added during the assay inhibited activities of both phosphatases with phosphorylase a and casein substrates. With histone as substrate, PPi slightly inhibited enzyme activities at lower concentrations (0.01-0.25 mM) but activated at higher concentrations. Pi activated both enzymes with this substrate; maximal activation being observed at a concentration of 5 mM.

Adenosine Diphosphate↗
PubMed1980

Purification and properties of phosphoprotein phosphatase from rabbit heart.

A low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been purified to homogeneity from rabbit heart by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34,000. The S20,w value and Stockes radius for the enzyme were 3.45 and 24.0 A, respectively. Using these two values, a molecular weight of 35,000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. A heat-stable protein inhibitor for this enzyme with phosphorylase a as the substrate was also shown to be present in crude extracts of rabbit heart. It inhibited the dephosphorylation of phosphorylase a by phosphoprotein phosphatase by decreasing the Vmax of the reaction.

Animals↗
PubMed1979

The properties of purified low molecular weight phosphoprotein phosphatase from rabbit heart.

A simplified procedure for the purification of low molecular weight phosphoprotein phosphatase acting on muscle phosphorylase a has been described from rabbit heart. The enzyme was purified to homogeneity by acid precipitation, ethanol treatment, and chromatography on Sephadex G-75 and Sepharose-histone. The purified enzyme showed a single band when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecular weight calculated by this method was 34 000. The S20, W value and Stokes radius for the enzyme was 3.35 and 24.0 A(1 A = 0.1 nm), respectively. Using these two values, a molecular weight of 35 000 was calculated. Purified enzyme showed a wide substrate specificity and catalyzed the dephosphorylation of phosphorylase a, glycogen synthase D, phosphorylated histone, and phosphorylated casein. Kinetic studies revealed the lowest Km with glycogen synthase D and maximum Vmax for the reaction with phosphorylase a.

Ammonium Sulfate↗
PubMed1978

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.

Animals↗
PubMed1977

The effect of streptozotocin-induced diabetes and of insulin supplementation on glycogen metabolism in rat liver.

The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.

Animals↗
PubMed1977

Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.

Animals↗
PubMed1977

Studies on the role of adenosine 3':5'-monophosphate in the activation of liver phosphorylase.

Crude extracts of rabbit liver, preincubated to promote the dephosphorylation of enzymes or other regulatory proteins, were used to study the role of cyclic AMP in the activation of glycogen phosphorylase. Inasmuch as endogenous liver phosphorylase was irreversibly altered by the preincubation procedure, crystalline skeletal muscle phosphorylase was used as the substrate in these studies. In the presence of magnesium ions and ATP, phosphorylase b was converted to phosphorylase a, and in an apparent biphasic process the phosphorylase a formed was subsequently converted to phosphorylase b. In the presence of adenosine 3':5'-monophosphate the rate of phosphorylase a formation and the maximal amount of phosphorylase a formed were increased. The cyclic AMP effect was enhanced by glucose-6-P and required the presence of glycogen. The catalytic subunit of cyclic AMP-dependent protein kinase could replace cyclic AMP in the stimulation of phosphorylase a formation. The effects of cyclic AMP or the catalytic subunit were shown to be due to stimulation of phosphorylase kinase rather than to inhibition of phosphorylase phosphatase. Preliminary fractionation experiments showed that it is possible to separate phosphorylase kinase catalytic activity from a factor or factors required for stimulation of its activation by the catalytic subunit.

Animals↗