Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes. Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

Adenylyl Cyclases↗

PubMed1986

GTP-activated GTP binding protein(Gs) in membranes achieved by hormone plus GDP does not serve as a substrate for ADP-ribosylation by cholera toxin.

Adenylate cyclase in the presence of GTP became active by the addition of cholera toxin irrespective of the presence of glucagon, and under the same condition the Gs of these activated enzymes were good acceptor of an ADP-ribose moiety. On the other hand, the cyclase in the presence of GDP remained inactive with cholera toxin but became active by the further addition of glucagon. However, neither of these Gs served as a cholera toxin substrate. Glucagon reduced an inhibitory action of added GDP for cholera toxin plus GTP-stimulated adenylate cyclase activity but did not for toxin plus GTP-enhanced ADP-ribosylation of Gs. These results demonstrate that Gs-GTP complex formation alone is not sufficient for Gs to serve as a cholera toxin substrate, and suggest an additional GTP binding site responsible for ADP-ribosylation by the toxin. Hormone dependent preferential interaction between the GTP binding site on Gs coupled with adenylate cyclase regulation and membrane-associated nucleoside diphosphate kinase is discussed.

Adenosine Diphosphate Ribose↗

PubMed1986

A rapid enzyme immunoassay for measurement of HBK in blood.

An enzyme immunoassay has been developed for the measurement of HBK (4-amino-2-hydroxybutylyldibekacin) a new semisynthetic aminoglycoside antibiotic. Antisera were raised in rabbits by immunization with HBK conjugated to bovine serum albumin (BSA). 3'-Eno-HBK conjugated to alkaline phosphatase (ALP) was used as an enzyme labeled antigen. The antibody-bound drug was separated from free using goat anti-rabbit IgG serum. The assay can be completed within one hr by co-incubating the first and the second antibody. The present immunoassay allows detection of 10 ng HBK per ml of serum, and is applicable for monitoring HBK level in blood. HBK concentrations in human sera were determined by the immunoassay during and after infusion and the levels were compared with those determined by high performance liquid chromatography (HPLC) and antimicrobial assay.

Aminoglycosides↗

PubMed1986