Characterization of 19-nor-10-oxo-25-hydroxyvitamin D3 production by solubilized chick kidney mitochondria and bovine serum albumin.
The vitamin D3 metabolite obtained from the incubation of 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)-solubilized chick kidney mitochondria with 25-hydroxyvitamin D3 (25-OH-D3) was identified to be 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3 (5(E)-19-nor). The production of 19-nor was dependent on time and on protein concentration, but was not dependent on the pH of the incubation. 19-Nor was not formed in the absence of protein or when protein had been heat-treated following detergent solubilization. 19-Nor was not further metabolized to any other product upon incubation with the CHAPSO-solubilized proteins. No 19-nor-10-oxo derivative of 1,25(OH)2D3 was formed when 1,25(OH)2D3 was used as substrate in the incubation. Kinetic analysis showed a substrate saturation with an apparent Vmax of about 4.1 pmol/min.mg and S0.5 of approximately 1.3 x 10(-6) M. The production of 19-nor was not restricted to the CHAPSO-soluble protein fraction of kidney mitochondria but was also found in both the CHAPSO-soluble and -insoluble fractions of chick liver mitochondria and CHAPSO-treated bovine serum albumin (BSA). 19-Nor production by detergent-treated BSA also showed saturation kinetics with a similar S0.5 and an apparent Vmax which was about 5-fold higher than that obtained with CHAPSO-solubilized mitochondria. The evidence suggests that the formation of 19-nor is not mediated by a traditional enzyme, but does require protein. A mechanism for the conversion of 25-OH-E3 to 19-nor is proposed, in which the naturally-occurring 5(Z)-25-OH-D3 substrate binds to protein, isomerizes to 5(E)-25-OH-D3 and is oxidized by hydrogen peroxide to 5(E)-19-nor via a dioxetane intermediate.
Department of Biochemistry, University of California, Riverside 92521.
J M
JM
Tran
H L
HL
Henry
D000818:Animals; D002112:Calcifediol / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002645:Chickens; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism; D010100:Oxygen; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
D002793:Cholic Acids; D003902:Detergents; D011506:Proteins; D012710:Serum Albumin, Bovine; C048531:chapso; C039030:19-nor-10-keto-25-hydroxyvitamin D3; D002112:Calcifediol; D010100:Oxygen