Jay M. McDonald

A sensitive and precise isotopic assay of ATPase activity.

Abstract A manual ATPase assay which measures the release of 32Pi from [γ-32P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate 32Pi from [γ-32P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10−15 to 10−7 mol of 32Pi from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-32P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.

Preliminary Characterization of Calcium Binding in Islet-cell Plasma Membranes

SummaryPreliminary characterization of calcium binding was determined in a highly-enriched islet-cell plasma membrane fraction using a membrane filtration technique. Equilibrium calcium binding was specific, concentration dependent and saturable. Scatchard analysis indicated the existence of more than one class of calcium binding sites. The affinity constants and maximum binding capacities were 1.14 ×105 M-1 and 1.2 picomol/μg protein and 1.17× 103 M-1 and 64.8 picomol/μg for the high and low affinity sites, respectively. Bound 45Ca2+ was dissociated from the plasma membranes in a biphasic manner in the presence of excess unlabelled calcium.

The ability of insulin to alter the stable calcium pools of isolated adipocyte subcellular fractions.

Abstract The calcium contents of adipocyte plasma membranes, mitochondria and microsomes (endoplasmic reticulum) were measured by atomic absorption. Ruthenium red (RR) and EDTA were used to selectively modify post-homogenization artifacts. Values obtained in the presence of RR appear to approximate the in vivo calcium content for mitochondria. Measurements in the presence of EDTA represented a highly stable pool of calcium for all organelles. Insulin (100 micro-units/ml) treatment of adipocytes prior to fractionation caused a conversion of a significant portion of the stable calcium pool to an exchangeable form in mitochondria without altering the total mitochondrial calcium content. Insulin did not affect the calcium content of the plasma membranes. The hormone did significantly increase the calcium content of the microsomes, which probably reflected the increased ability of microsomes isolated from insulin-treated adipocytes to bind and accumulate calcium.

Depression increases risk of incident myocardial infarction among Veterans Administration patients with rheumatoid arthritis

OBJECTIVE This study evaluates whether depression is a risk factor for incident myocardial infarction (MI) in Department of Veterans Affairs (VA) patients with rheumatoid arthritis (RA) between 30 and 79 years of age. METHODS We used a retrospective cohort study of 15,634 patients with RA. Diagnoses and sociodemographic data were obtained from VA administrative and pharmacy databases between fiscal years 1999 and 2006. Entry into the cohort required 2 years of patient time with no evidence of cardiovascular disease. Cox proportional hazard models with time-dependent covariates were computed to determine whether RA patients with depression as compared to RA patients without depression were at increased risk for MI during the maximum 6-year follow-up period. RESULTS Unadjusted analyses indicated depressed RA patients were 1.4 times more likely than nondepressed RA patients to have an MI during follow-up. These results remained significant (HR=1.4; 95% CI: 1.1-1.8) in the adjusted Cox proportional hazards model which included the effects of sociodemographics and known physical risks (e.g., diabetes) for MI. CONCLUSIONS Depressed RA patients, without a history of cardiovascular disease, are 40% more likely to have a heart attack as compared to those without depression. These data demonstrate a rapid (within 6 years) transition to MI following onset of depression in RA patients. Increased monitoring of depression and heart disease status in this patient population may be warranted which in turn may result in longer duration of life.

Calmodulin-activated protein kinase activity in rat pancreatic islet cell membranes

Abstract A calmodulin-activated protein kinase activity was identified in pancreatic islet cell membranes, which phosphorylated an endogenous protein of molecular weight 57,000 as determined by sodium dodecyl sulfate electrophoresis. Calmodulin activation of the protein kinase activity was dose dependent and saturable, with half-maximal activation occurring at 360 n m calmodulin. Trifluoperazine inhibited calmodulin activation of the protein kinase but had negligible effects on basal activity. The 50% inhibition occurred at 40 μ m trifluoperazine. Subcellular fractionation of islet cells demonstrated that the calmodulin-activated activity was enriched in a light-particle fraction. This fraction was enriched in endoplasmic reticulum, but there was no correlation between protein kinase activity and endoplasmic reticulum marker enzyme activity among various subcellular fractions. The function of the kinase activity and the identity of the substrate are unknown, but the kinase activity may be involved in pancreatic stimulus-secretion coupling.

Parallel stimulation in adipocytes of the plasma membrane Ca2+-transport/(Ca2+ + MG2+)-ATPase system and mitochondrial pyruvate dehydrogenase by a supernatant factor derived from isolated plasma membranesi

Summary A factor derived from the supernatant of adipocyte plasma membranes which mimics the effects of insulin on mitochondrial pyruvate dehydrogenase (Kiechle, F.L., Jarett, L., Kotagal, N., and Popp, D.A. (1981) J. Biol. Chem. 256, 2945–2951) was shown to activate a high affinity (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ transport of adipocyte plasma membranes. Fractionation of the supernatant by G25 Sephadex chromatography separated the material into four inactive and one active fraction. All of the fractions behaved identically in terms of activation of pyruvate dehydrogenase, (Ca2+ + Mg2+)- ATPase, and Ca2+ transport, suggesting that the active fraction responsible for stimulating mitochondrial pyruvate dehydrogenase was also responsible for stimulating the plasma membrane Ca2+-dependent processes.

The effects of stasis with and without exercise on free calcium, various cations, and related parameters.

The changes in free calcium, total calcium, albumin, protein, pH, lactate, sodium, magnesium, and potassium values during and following venous stasis (3 min at an external pressure of 100 mmHg) with and without forearm exercise have been measured. The pattern of changes observed with time were dependent on the presence or absence of forearm exercise. Without exercise significant changes are only observed at 1 min following stasis and then the increases are only moderate (free calcium 2.0%, total calcium 2.4%, albumin 6.9%, and protein 5.9%). However, when the forearm is exercised, larger increases (free calcium 8.6%, total calcium 8.4%, albumin and protein 12.4%) were observed for all parameters following stasis. These increases took 1 to 3 min to return to baseline for most parameters. Only potassium and magnesium went below baseline during the recovery period. It is recommended that when a tourniquet is used to aid in obtaining venous blood samples for these analytes, exercise of the forearm be avoided. If forearm exercise is unavoidable, then the sample should be obtained 1 to 3 min after release of the tourniquet.

Evidence that protein kinase C is involved in regulating glucose transport in the adipocyte

The role of protein kinase C in the mechanism of stimulation of glucose transport in rat adipocytes was investigated. Glucose transport was stimulated by dioleoylglycerol (DOG), tetradecanoyl phorbol acetate (TPA) and phospholipase C (PLC). Agents that inhibit protein kinase C (polymyxin B, gossypol and quercitin) also inhibited glucose transport that had been stimulated by DOG, TPA, PLC and insulin.

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum.

Abstract The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum ( D. E. Bruns, J. M. McDonald, and L. Jarett, 1976 , J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca2+ + Mg2+-ATPase) of relatively low activity. Mg2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca2+ + Mg2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent Km values of 115 μ m for ATP, 333 μ m for magnesium, and 200 μ m for calcium. Maximum Mg2+-ATPase activity was obtained with no added calcium and 1 m m magnesium. Potassium was found to inhibit Mg2+-ATPase activity at concentrations greater than 100 m m . The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μ m , respectively. (Ca2+ + Mg2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 m m and maximum activity reached at 100 m m potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase are similar to those of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.