Comparison of bone and osteosarcoma adenylate cyclase. Partial purification of membranes and kinetic properties of enzyme

Abstract

The purpose of this study was to compare the adenylate cyclase of a tumour (rat osteosarcoma) growing in vivo with that of fast-growing embryonic bone. In the tumour the enzyme activity per total protein or DNA (under the same assay conditions) was 6--10-fold lower than in embryonic bone. To characterize this difference, we examined the kinetic properties of the enzyme in partially purified plasma membranes from the two tissues. A purification procedure based on differential centrifugation and discontinuous-sucrose-gradient centrifugation yielded a 10-fold increase in the specific activities of adenylate cyclase and 5'-nucleotidase in bone. The same procedure yielded an enriched membrane preparation from the tumour, but, relative to 5'-nucleotidase, a loss of 30% in adenylate cyclase occurred, which could not be recovered from another fraction. Kinetic analysis revealed that the lower adenylate cyclase activity in the tumour was due to a decrease in Vmax.. There was no significant difference in Ks (approx. 0.15 mM), and in the Km for GTP and p[NH]ppG. There were marked differences, however, in the extent of stimulation by p[NH]ppG, GTP and hormone, which was greater in tumour, and in the K1 for adenosine inhibition, which was 140 microM in bone and 500 microM in tumour. Under maximum stimulatory conditions, the enzyme activity in the tumour approached that in bone. The kinetic differences between bone and tumour enzyme were decreased by detergent solubilization, suggesting that the membrane environment plays a role in the generation of the observed differences.