Comparison of bone and osteosarcoma adenylate cyclase. Partial purification of membranes and kinetic properties of enzyme
- 1 March 1980
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 185 (3), 617-627
- https://doi.org/10.1042/bj1850617
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.This publication has 39 references indexed in Scilit:
- Interrelationship Between Adenylate Cyclase Activity, Adenosine 3′:5′ Cyclic Monophosphate Phosphodiesterase Activity, Adenosine 3′:5′ Cyclic Monophosphate Levels, and Growth of Cells in CultureProceedings of the National Academy of Sciences, 1973
- Alteration of Cell-Surface Proteins by Viral Transformation and by ProteolysisProceedings of the National Academy of Sciences, 1973
- Transformation of Chick-Embryo Fibroblasts by Wild-Type and Temperature-Sensitive Rous Sarcoma Virus Alters Adenylate Cyclase ActivityProceedings of the National Academy of Sciences, 1973
- Adenyl cyclase.1973
- Growth Control and Cyclic Alterations of Cyclic AMP in the Cell CycleNature New Biology, 1972
- A Mutation in a Rous Sarcoma Virus Gene That Controls Adenosine 3′,5′-Monophosphate Levels and TransformationJournal of Biological Chemistry, 1972
- Composition and Synthesis of Gangliosides in Rat Hepatocyte and Hepatoma Cell LinesJournal of Biological Chemistry, 1969
- INTERACTION OF THE CARBOHYDRATE-BINDING PROTEIN CONCANAVALIN A WITH NORMAL AND TRANSFORMED CELLSProceedings of the National Academy of Sciences, 1969
- Carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. I. Glucosamine-labeling patterns in 3T3, spontaneously transformed 3T3, and SV-40-transformed 3T3 cellsBiochemistry, 1969
- A DIFFERENCE IN THE ARCHITECTURE OF THE SURFACE MEMBRANE OF NORMAL AND VIRALLY TRANSFORMED CELLSProceedings of the National Academy of Sciences, 1969