Biomedical subjects

J F Krall

Publications and source records attributed to J F Krall.

At least 37 records · Page 2Linked to original sources

PubMed1983

Lymphocyte adenylate cyclase and human aging.

Adenylate cyclase activity was determined by enzymatic conversion of [32P]ATP to [32P]cAMP using peripheral lymphocytes freshly isolated from human subjects. The lymphocyte enzyme was stimulated by the potent beta-adrenergic catecholamine agonist isoproterenol and by the nonhydrolyzable GTP-analog Gpp[NH]p. The two activators had a synergistic effect, and agonist-dependent enzyme activity followed simple Michaelis-Menten kinetics with respect to isoproterenol in the presence but not in the absence of Gpp[NH]p. Cyclic AMP production by intact lymphocytes, determined by protein binding assay, also followed simple Michaelis-Menten kinetics with respect to isoproterenol. Kact of isoproterenol was the same in intact cells and the broken cell assay in the presence of Gpp[NH]p, suggesting the indispensable role the GTP-binding coupling factors play in the intact lymphocyte. In 31 human subjects between the age of 21 and 103, adenylate cyclase activity in the presence of isoproterenol, Gpp[NH]p, or isoproterenol in the presence of Gpp[NH]p decreased with the increasing age of the subject. The sensitivity of the enzyme to stimulation by isoproterenol, defined as the Kact and determined in the presence of Gpp[NH]p, was the same in lymphocytes from young (less than 45 years) or elderly (greater than 75 years) subjects. These results suggest a deficiency in the lymphocyte adenylate cyclase system distal to the beta-adrenergic catecholamine receptor could account for deterioration of cAMP-mediated components of the immune response which occur with age.

Adenylyl Cyclases↗

PubMed1982

Adenylate cyclase activation. Characterization of guanyl nucleotide requirements by direct radioligand-binding methods.

Particulate adenylate cyclase preparations from rat uterine smooth muscle had a single class of [3H]guanyl-5'-yl imidodiphosphate ([3H]GMP.P(NH)P)-binding sites with all of the properties of the guanyl nucleotide-requiring enzyme activation sites (N) which couple hormone receptors and catalytic subunits. These sites bound the radioligand in a reversible manner at low temperature (less than 2 degrees C) but irreversibly at temperatures between 6 and 24 degrees C, properties characteristic of the activation of the enzyme by treatment with GMP.P(NH)P described previously (Krall, J. F., Leshon, S. C., Frolich, M., and Korenman, S. G. (1981) J. Biol. Chem. 256, 5436-5442). Temperature affected the number but not the apparent affinity (Kd approximately 1.0 microM) of binding. The time course of the transition from reversible to irreversible binding was coincident with the irreversible activation of the catalytic subunit. The methyl analog of GTP, guanyl-(beta, gamma-methylene)-diphosphate, a poor irreversible activator compared to GMP.P(NH)P in this enzyme system, was a competitive inhibitor of [3H]GMP.P(NH)P binding but with a 10-fold lower affinity (Kd approximately 10 microM). Using these direct radioligand-binding methods, both an inactive (NL) and active (NL*) form of the guanyl nucleotide-binding activation site were demonstrated, and the transition NL leads to NL* was identified as the temperature-dependent event in catalytic subunit (C) activation. The nondissociability of specific [3H]GMP.P(NH)P binding and the irreversible nature of catalytic subunit activation suggest that a complex of NL*.C characterizes the fully activated state of the smooth muscle enzyme.

Adenylyl Cyclases↗

PubMed1982

Kinetics and thermodynamics of activation of pretreatment with guanosine 5'-[beta, gamma-imido]triphosphate of smooth-muscle adenylate cyclase.

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2 degrees C. Activation (C-->C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37 degrees C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37 degrees C. Over the range 6-24 degrees C, activation by pretreatment with p[NH]ppG followed simple Michaelis-Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K(m) for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37 degrees C) was moderately endergonic (DeltaH(0) approximately 8kJ.mol(-1)) and accompanied by an increase in entropy (DeltaS(0) approximately 146J.mol(-1).K(-1)). The beta-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K(m) at every temperature. This result suggested that formation of the receptor-hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the beta-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.

Adenylyl Cyclases↗

PubMed1982