Ramji L. Khandelwal

Some properties of purified phosphoprotein phophatases 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.

Solubilization of a calcium dependent adenosine triphosphatase from rat heart sarcolemma

Abstract The rat heart sarcolemmal ATPase activity was found to be dependent on the presence of Ca 2+ ( v max = 37 μ mol Pi/mg protein/h and K a = 0.59 m m ) or Mg 2+ ions ( v max = 28 μ mol Pi/mg protein/h and K a = 0.87 m m ). The incubation of sarcolemmal membrane with trypsin stimulated the Ca 2+ dependent ATPase activity without affecting the Mg 2+ dependent ATPase activity. The increase in Ca 2+ dependent ATPase activity was associated with a decrease in the K a value from 0.59 to 0.45 m m and an increase in v max from 37 to 69 μmol Pi/mg protein/h. In membrane preparations treated with detergents, trypsin decreased the Mg 2+ dependent ATPase activity whereas the Ca 2+ dependent ATPase activity was increased. Trypsin treatment of sarcolemma released proteins in the supernatant that showed ATP hydrolysis in the presence of Ca 2+ but not Mg 2+ ; while the residual membrane showed an ATPase dependent on Ca 2+ ( v max = 67 μ mol Pi/mg protein/h and K a = 0.58 m m ) and Mg 2+ ( v max = 58 μ mol Pi/mg protein/h and K a = 0.87 m m ). The proteins released in the supernatant were subjected to column chromatography on sepharose-6B, and DEAE cellulose and a Ca 2+ -dependent ATPase activity ( v max = 200 μ mol Pi/mg protein/h and K a = 0.25 m m ) was recovered as a distinct peak. These results indicate solubilization of a Ca 2+ dependent ATPase which, unlike the enzyme present in heart sarcolemma, showed negligible activation by Mg 2+ .

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system

Abstract The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.

Activation of heart sarcolemmal Ca2+Mg2+ ATPase by cyclic AMP-dependent protein kinase

Abstract The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate 32P from [γ-32P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca2+ ATPase and Mg2+ ATPase activities without any changes in Na+K+ ATPase activity. The observed increase in Ca 2+ Mg 2+ ATPase activity was found to be associated with an increase in Vmax value of the reaction whereas Ka value for Ca 2+ Mg 2+ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.

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.

Studies on inactivation and reactivation of homogeneous rabbit liver phosphoprotein phosphatases by inorganic pyrophosphate 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.

The regulation of liver phosphoprotein phosphatase by inorganic pyrophosphate and cobalt.

Abstract The preincubation of rat liver crude extracts with ATP caused a 60% inactivation of phosphoprotein phosphatase in 30 min at 30 °C. The presence of Mg2+, or cyclic AMP, along with ATP in the preincubation mixture had no effect on the inactivation of phosphatase caused by ATP. The crude liver phosphatase was also inactivated by ADP or PPi; PPi being the most potent inactivating metabolite. AMP, adenosine or Pi were without any effect. The effect of ATP or PPi was completely reversed by cobalt. The cobalt effect was very specific and could not be replaced by several metal ions tested except by Mn2+ which was partly active. With the aid of sucrose density gradient studies, it was also shown that PPicauses an apparent conversion of a 4.1 S form to a 7.8 S form of the enzyme in rat liver extracts. Cobalt, on the other hand, converts the higher 7.8 S form to a lower 4.1 S form of the enzyme. The preincubation of purified rabbit liver phosphoprotein phosphatase with PPi also caused a complete inactivation of the enzyme in 40 min. The inactivation of the enzyme by PPi was completely reversed by cobalt. Unlike the apparent interconversion between different molecular forms of the enzyme by PPi and cobalt in rat liver crude extracts, no such interconversion of purified rabbit liver phosphoprotein phosphatase was observed in the presence of PPi and cobalt.

Protein kinase activity in cariogenic and non-cariogenic oral streptococci: Activation and inhibition by cyclic AMP

In recent years, considerable information has been obtained concerning the activity of cyclic AMP-dependent protein kinases in various mammalian tissues and in a number of other animal phyla [ 1,2], with this activity being associated both with particulate and soluble cellular fractions [3-9] . However, despite this interest, only two microorganisms, E. coli [lo] and the acellular slime mold, Physarum polycephalum [ 1 l] , have been shown to contain protein kinases influenced by cyclic AMP. On the one hand, histone phosphorylation by the E. coli enzyme was enhanced 3fold by cyclic AMP, while P. polycephalum was shown to contain both a cyclic AMP-activated protein kinase and a cyclic AMP-inhibitable enzyme. We have previously reported that the oral microbe, Streptococcus salivarius, synthesizes cyclic AMP through the action of multiple adenyl cyclases [ 121 . Subsequent study with highly purified adenyl cyclase III showed that physiological concentrations of various cellular metabolites regulated cyclic AMP formation in vitro [ 131 . Since little is known concerning the effect of cyclic AMP on protein phosphorylation in bacteria, we undertook to determine whether protein kinase activity, as well as cyclic AMP-binding protein, were present in S. salivarius, S. sanguis and various strains of the cariogenic genus, S. mutans. We wish to report the presence of cyclic AMP-activated protein kinase activity and cyclic AMP-binding protein in these bacteria. Furthermore, since several strains were shown to contain both cyclic AMP-inhibitable as well as cyclic AMP-activated protein kinase activity, these bacteria probably contain at least two protein kinases regulated in a reciprocal fashion. 246 2. Materials and methods

Effectors of purified adenyl cyclase from Streptococcus salivarius

Abstract The activity of highly purified (3200-fold) adenyl cyclase from Streptococcus salivarius was examined in the presence and absence of P i , inorganic pyrophosphate, nucleotides and glycolytic intermediates. The enzyme was inhibited by P i , ADP and all the triphosphates and monophosphates of guanosine, uridine, inosine and cytidine; inhibition was completely competitive. The inhibition in the presence of PP i was partially competitive, while AMP produced inhibition of the mixed type, i.e., partially competitive and completely noncompetitive. PP i was the most potent inhibitor ( K i = 0.23 m m ) followed by ADP ( K i = 0.43 m m ), GTP ( K i = 0.52 m m ) and UTP ( K i = 0.60 m m ). Severe inhibition of the enzyme was also observed in the presence of the diphosphate and sugar nucleotides of the above bases at concentrations between 0.1 and 5 m m , when tested at one substrate concentration (ATP = 0.6 m m ). The respective cyclic 3′,5′-nucleoside monophosphates were, however, only slightly inhibitory (maximum 11%). While the nucleotides were generally inhibitory, the activity of the enzyme was variable in the presence of various glycolytic intermediates. Weak activation of adenyl cyclase activity was observed with glucose-6-P, glucose-1-P, 2-P-glycerate and pyruvate. 2-P-glycerate required the lowest concentration for half maximal activation ( K a = 0.13 m m ) followed by glucose-1-P (0.24 m m ), glucose-6-P (0.55 m m ) and pyruvate (1.12 m m ). These compounds increased the V of the enzyme without affecting the apparent K m for ATP. Fructose-6-P, fructose-1,6-P 2 , glyceraldehyde-3-P and P-enolpyruvate inhibited or activated the enzyme depending upon the concentration of the compound used. Both the apparent K m for ATP as well as the V were altered by fructose-6-P and fructose-1,6-P 2 . However, activating concentrations of glyceraldehyde-3-P and P-enolpyruvate increased the V without affecting the apparent K m , whereas inhibiting concentrations decreased the V and increased the K m for the substrate.

Fluoride inhibition of glucose-6-P formation in Streptococcus salivarius: Relation to glycogen synthesis and degradation

Abstract The concentrations of glucose-6-P and ATP in intact cells of the oral microbe, Streptococcus salivarius , were analyzed during anaerobic glucose metabolism in the presence and absence of fluoride. Addition of 2.4 m m NaF to cells actively degrading glucose resulted in an immediate decrease in the cellular glucose-6-P and ATP content concomitant with the complete inhibition of glucose uptake and glycogen synthesis. A noticeable decrease in cellular glucose-6-P concentration was produced in cells metabolizing glucose by concentrations of NaF as low as 0.06 m m , regardless of whether the inhibitor was added before or after the substrate. After the initial decline in the glucose-6-P content, the concentration of this intermediate frequently increased, resulting from the degradation of endogenous glycogen. ATP, on the other hand, remained low throughout the experimental period. Experiments with crude enzyme preparations of the organism demonstrated that the glycogen synthetic enzymes, phosphoglucomutase (EC 2.7.5.1), ADPG pyrophosphorylase, and ADPG glucan transferase (ADP-glucose:glycogen glucosyl transferase), as well as phosphorylase (EC 2.4.1.1) in the degradative pathway, were fluoride-insensitive at NaF concentrations inhibiting in vivo synthesis. The inability of 2.4 m m NaF to inhibit glucose-6-P formation from glycogen in vivo in the absence of exogenous glucose confirms that phosphoglucomutase and phosphorylase in this organism are not inhibited by fluoride. The results strongly suggest that fluoride interacts, in some manner, with the sugar transport system in the organism as phosphorylation by hexokinase (EC 2.7.1.1) was not fluoride-sensitive. The “apparent” inhibition of glycogen synthesis is discussed in relation to the availability of ATP and glucose-6-P.