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R L Khandelwal

Publications and source records attributed to R L Khandelwal.

At least 55 records · Page 3Linked to original sources
PubMed1986

The diurnal rhythm of liver glycogen phosphorylase: correlating changes in enzyme activity and enzymic protein.

The diurnal rhythm of phosphorylase activity in mouse liver extracts was correlated with the 24 h fluctuations in phosphorylase protein. This last measurement was made using rocket immunoelectrophoresis. The peak activity of phosphorylase appeared coincident (at 18:00 h) with the greatest amount of phosphorylase protein detected. Conversely, the lowest activity measured and lowest enzymic protein content both occurred at 02:00 h. Regression analysis revealed a significant positive correlation between enzyme activity and protein. Thus changes in the cellular concentration of this enzyme are implicated in the diurnal rhythm of liver glycogen.

Animals↗
PubMed1986

Glycogen synthetic abilities of Actinomyces viscosus and Actinomyces naeslundii freshly isolated from dental plaque over root surface caries lesions and non-carious sites.

Relative glycogen synthetic abilities of resting cells of fresh clinical isolates of Actinomyces viscosus and Actinomyces naeslundii originating from dental plaque samples over root surface caries lesions and non-carious sites were studied under anaerobic conditions at a constant pH of 7.0, with U-(14C)-glucose used as the carbon source. Although the rates of glucose utilization and total acid formation were essentially the same, A. viscosus strains isolated from root surface caries lesions showed glycogen synthetic abilities approximately two to seven times higher than did A. viscosus strains originating from non-carious sites, and also two to four times higher than did A. naeslundii strains originating from both carious and non-carious sites.

Acids↗
PubMed1986

Kinetic properties of glycogen synthase and phosphorylase and structural aspects of glycogen in the db/db mouse liver.

Kinetic studies were carried out on liver glycogen synthase and phosphorylase isolated from genetically diabetic db/db mice. Glycogen synthase a and b enzymes from diabetic mice had Vmax values 30% and 20% lower, respectively, than the enzymes from normal mice. Glycogen synthase b from diabetic mice also had a 30% lower I0.5 for Pi and ATP at physiologic concentrations of UDP-glucose (0.25 mM) compared with the normal enzyme. Kinetic studies of phosphorylase a showed that, at low glycogen concentrations (0.25 mg/ml), the Vmax of the diabetic enzyme was twofold greater than that of the normal enzyme. This was probably related to the diabetic phosphorylase a having a lower apparent Km for glycogen. This enzyme also had a slightly higher I0.5 for ATP compared with the enzyme from normal mice. Structural studies of liver glycogen isolated from these diabetic mice showed differences from normal mouse glycogen. Both the alpha- and beta-amylase limits were lower in the diabetic glycogen, and the average chain lengths, exterior chain lengths, and interior chain lengths calculated from these limits were all shorter in the glycogen from diabetic mice. Although both normal and diabetic glycogen absorbed light maximally at 430 nm when complexed with iodine, the absolute absorbance value was significantly lower for the diabetic glycogen. These data suggest an altered branching pattern of liver glycogen from the diabetic mice and it is suggested that this altered structure may ultimately influence the activities of glycogen-metabolizing enzymes. These results provide further characterization of the db/db mouse and show heretofore undescribed changes in phosphorylase a kinetics and glycogen structure that occur in diabetes.

Animals↗
PubMed1985

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver.

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.

Animals↗
PubMed1985

Resolution of bovine brain calcineurin subunits: stimulatory effect of subunit B on subunit A phosphatase activity.

Calcineurin was dissociated into subunits A and B by SDS and the dissociated subunits were separated by Sephadex G-100 column chromatography in SDS. The phosphatase activity was associated with the A subunit and was detected only in the presence of MnCl2 of the various divalent cations tested. The Mn2+-dependent phosphatase of A subunit was stimulated (4-5-fold) by calmodulin. The subunit B increased only modestly Mn2+ stimulated phosphatase activity of subunit A but markedly increased it when assay also contained calmodulin. These results support the view that subunit B plays an important role in Mn2+/calmodulin regulation of subunit A phosphatase activity. They also lend further support to our earlier postulate ([1984] FEBS Lett. 169, 251-255) that Mn2+ is a powerful regulator of calcineurin phosphatase.

Animals↗
PubMed1985

Diurnal variations in the activities of the glycogen metabolizing enzymes in mouse liver.

The glycogen level in mouse liver was maximal during the night and decreased to the lowest level during the light period. The peak activity of phosphorylase alpha was observed during the light hours and thus paralleled the decline of hepatic glycogen concentrations. The period of rapid glycogen synthesis (1800-2200 hr) was immediately preceded by maximum glycogen synthase alpha activity. Significant diurnal rhythms for phosphorylase kinase and phosphorylase phosphatase activities were also observed and appear to play a role in regulating the diurnal rhythm of phosphorylase alpha activity.

Animals↗
PubMed1985

Aberrations in the diurnal rhythms of plasma glucose, plasma insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities in genetically diabetic (db/db) mice.

The diurnal rhythms of plasma glucose, insulin, liver glycogen, and hepatic glycogen synthase and phosphorylase activities were determined in control and genetically diabetic (db/db) mice 8 weeks of age. The diabetic mice showed wide fluctuations in their plasma glucose levels, although being similar to controls near the end of the light period. Little variation was observed in their elevated plasma insulin levels. Liver glycogen levels in diabetic mice were not depleted to the low levels seen in controls during the last part of the light period but were maintained at significantly higher levels. However, maximum attained glycogen levels were similar in the two groups of mice. Alterations were also observed for the diurnal rhythms of glycogen synthase and phosphorylase activities, although again the daily maximums were similar in control and diabetic mice. These findings suggest that the reported changes of several of these metabolic parameters in the db/db mouse may be due to alterations in the diurnal pattern rather than to absolute changes.

Animals↗
PubMed1985

Effect of weaning on the activities of glycogen synthase and phosphorylase in rat liver.

The effects of weaning on the level of glycogen and the activities of glycogen synthase and phosphorylase were determined in rat liver. Glycogen levels in rat liver increased at the start of the weaning period and reached a plateau on postnatal day 20. The active form of glycogen synthase increased until postnatal day 19 and then declined. Total glycogen synthase (active + inactive) remained high during the suckling period and declined to a new low level during the weaning period. The activity ratio (active/total) increased from day 16 to days 18-22 and then decreased to the same level as found during the suckling period. At the onset of weaning the active form of phosphorylase decreased, whereas total phosphorylase initially increased and then decreased after postnatal day 20. Both forms of phosphorylase increased again at the end of the weaning period. The activity ratio decreased at the start of weaning and remained low throughout the rest of the weaning period. The effects of premature weaning were similar to those observed in normally weaned animals, but the changes occurred sooner and were more pronounced.

Aging↗
PubMed1985

Isolation and characterization of calcineurin from bovine brain.

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Microsomal phosphorylase in rat heart: depletion following adrenalectomy and restoration by in vivo administration of dexamethasone.

Phosphorylase activities were determined in homogenates, particulate and soluble fractions of heart muscle from control, adrenalectomized, and adrenalectomized dexamethasone-treated rats so as to assess the influence, if any, of glucocorticoids on the subcellular distribution of this enzyme. The specific activities (enzyme activity expressed as units per mg protein) and recoveries (enzyme activity expressed as units per fraction/g tissue) of both the active form of phosphorylase (phosphorylase a) and total phosphorylase (phosphorylases a and b) were essentially similar in homogenates and 10,000 X g particulate fractions prepared from hearts of control, adrenalectomized, and adrenalectomized dexamethasone-treated rats. Interestingly, however, the specific activities and recoveries of active as well as total phosphorylase were markedly lower (70-80%; P less than 0.001) in cardiac microsomes of adrenalectomized compared to control rats. On the other hand, the specific activities and recoveries of phosphorylase (active and total) were significantly higher (30-60%; P less than 0.01 or 0.001) in the cytosol fraction of heart muscle from adrenalectomized compared to control rats. Treatment of adrenalectomized rats with dexamethasone (a synthetic glucocorticoid) restored the specific activities and recoveries of phosphorylase in heart microsomal and cytosol fractions to control levels. No appreciable differences were observed in the specific activity ratios of phosphorylase a to phosphorylases a and b in any of the subcellular fractions of cardiac muscle from control, adrenalectomized, and adrenalectomized dexamethasone-treated rats. The above findings suggest a potential involvement of glucocorticoids in the maintenance of the membrane-associated (microsomal) pool of phosphorylase and, therefore, in the cellular compartmentalization of this enzyme in heart muscle.

Adenosine Triphosphate↗
PubMed1985

Age-related changes in hepatic glycogen metabolism in the genetically diabetic (db/db) mouse.

Hepatic glycogen metabolism was investigated in genetically diabetic C57BL/KsJ-db/db mice during their development. Initially, the development of obesity, hyperglycemia, hyperinsulinemia, and hyperglucagonemia in these mice was examined, which illustrated that the diabetes progressed normally. Little difference in hepatic glycogen concentrations was observed, averaging approximately 50 and 60 mg/g liver in diabetic (db/db) and control heterozygote (db/+) mice, respectively. Glycogen synthase activity (total and a-form) was significantly elevated by 5 wk in the diabetic mice relative to controls and reached maximum levels (two-fold higher than controls) around 8-9 wk. This activity then slowly declined during the rest of the 15-wk period examined. Both phosphorylase a and total phosphorylase activities were also elevated by 5 wk, reaching levels twofold higher than controls. These activities did not decline at the end of this 15-wk period, but instead continued to slowly increase. Glycogen synthase a activity showed a positive correlation (r = 0.54, N = 144) with circulating levels of insulin, and a similar correlation was seen for phosphorylase a activity and plasma glucagon levels (r = 0.64, N = 72). Protein kinase and phosphoprotein phosphatase activities were also measured, but no differences were detected between diabetic and control mice. This longitudinal study clarifies some of the changes in hepatic glycogen metabolism that occur during the progression of diabetes in the db/db mouse and indicates a role for circulating insulin and glucagon concentrations on the steady-state activities of glycogen synthase and phosphorylase, respectively.

Aging↗
PubMed1984

Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: use of the phosphoenolpyruvate:sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins.

Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.

Adenosine Triphosphate↗
PubMed1984

Intrinsic phosphatase activity of bovine brain calcineurin requires a tightly bound trace metal.

The divalent metal requirement of intrinsic phosphatase activity was investigated using native and trypsinized calcineurin. This was assessed by examining (1) the stimulation of the enzyme by various metals, (2) the inhibition of the enzyme activity by metal chelators (EDTA and EGTA), and (3) the restoration by various metals of the activity of the EDTA-inhibited calcineurin phosphatase. The results supported the view that a tightly bound trace metal is necessary for expression of the phosphatase activity of calcineurin and implicate Mn2+ as the tightly bound metal.

Animals↗
PubMed1984

Activation of phosphoenolpyruvate-dependent protein kinase by cytidine 5'-triphosphate in rat skeletal muscle.

The effects of nucleotides on phosphoenolpyruvate-dependent protein kinase in rat skeletal muscle were examined. Various ribonucleotide triphosphates were tested and the protein kinase reaction was maximally activated by cytidine 5'-triphosphate followed by uridine 5'-triphosphate and guanosine 5'-triphosphate. No activation by adenosine 5'-triphosphate was observed. Cytidine 5'-diphosphate also activated the reaction but to a significantly lesser extent. Cytidine 5'-monophosphate and cyclic cytidine 3',5'-monophosphate showed no effect whereas cytidine was slightly inhibitory.

Adenosine Triphosphate↗
PubMed1984

Effects of age on glycogen synthase and phosphorylase activities in rat liver.

The activities of glycogen synthase and phosphorylase were determined in homogenates and subcellular fractions of liver from young adult (6 months' old), adult (12 months' old) and aged (24 months' old) rats. The specific activities (enzyme activity expressed as units per mg protein) of the active form of glycogen synthase (synthase a) as well as of total synthase (synthase a + b) were significantly lower in liver homogenates of aged compared to young adult or adult rats. The age-associated decrease in the specific activity of synthase a was most marked in the 10 000 g and 100 000 g particulate fractions of liver. The specific activities of the active form of phosphorylase (phosphorylase a) as well as of total phosphorylase (phosphorylase a + b) were also significantly lower in liver homogenates of aged compared to young adult or adult animals. The age-related decrease in the specific activity of phosphorylase a was most pronounced in the 10 000 g particulate fraction of liver. Analysis of the specific activity ratios of glycogen synthase a/glycogen synthase a + b indicated a disproportionately greater age-related decrement in the active form of this enzyme. No such age-related difference was evident in the specific activity ratios of phosphorylase a/phosphorylase a + b. The age-associated decrease in the activities of glycogen synthase (a or a + b) and phosphorylase (a or a + b) was also discernible when the enzyme activities were expressed as units per g liver; this decrement in enzyme activity was reflected in all subcellular fractions examined. The levels of plasma insulin, plasma glucose and liver glycogen were not significantly different in rats of the three age groups used in these studies. The reduced activities of liver glycogen synthase and phosphorylase in aged rats is indicative of a likely diminution in the turnover of glycogen in liver during aging. Such an age-associated deficit in liver glycogen metabolism may contribute, at least in part, to the generally observed glucose intolerance upon aging.

Aging↗
PubMed1983

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle.

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.

Adenosine Triphosphate↗