Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes

• Published in: Biochimica et biophysica acta Vol. 915; no. 2; pp. 246 - 253
• Main Authors: GUENGERICH, F. P, CHURCHILL, P. F, JUNG, C. Y, FLEISCHER, S
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 24.09.1987; North-Holland
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cytochrome b Group - analysis; Cytochrome b Group - radiation effects; Cytochrome P-450 Enzyme System - analysis; Cytochrome P-450 Enzyme System - radiation effects; Cytochromes b5; Enzymes and enzyme inhibitors; Epoxide Hydrolases - analysis; Epoxide Hydrolases - radiation effects; Fundamental and applied biological sciences. Psychology; Immunoelectrophoresis; Macromolecular Substances; Male; Membrane Proteins - analysis; Membrane Proteins - radiation effects; Microsomes, Liver - analysis; Molecular Weight; NADH, NADPH Oxidoreductases - analysis; NADH, NADPH Oxidoreductases - radiation effects; NADPH-Ferrihemoprotein Reductase - analysis; NADPH-Ferrihemoprotein Reductase - radiation effects; Oxidoreductases; Rats; Rats, Inbred Strains; Biological inactivity; Rat; Isozyme; Liver; Rodentia; Phospholipid; Molecular interaction; Microsome; Epoxide hydrolase; Molecular weight; Aggregation; Vertebrata; Mammalia; Flavoprotein-linked monooxygenase; Molecular association; Membrane; Immunoelectrophoresis; Catalyst activity
• ISSN: 0006-3002; 1878-2434

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.