Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1989-6-20
pubmed:abstractText
Pig kidney mitochondrial 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities have been solubilized with cholate/Emulgen 911 and reconstituted with NADPH, ferredoxin reductase and ferredoxin. All three of these components are required for full catalytic activity of both enzymes. Both products were identified by co-chromatography with authentic metabolites on both normal and reverse-phase h.p.l.c. using solvent systems which were shown to separate 10-oxo-19-nor-25-hydroxyvitamin D3 from 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3]. In addition, periodate treatment of the 24,25-(OH)2-D3 product resulted in complete loss of the product as measured by protein-binding assay. Further purification by p-chloroamphetamine-Sepharose chromatography of a solubilized extract from a pig fed a normal diet increased the specific content of the cytochrome P-450 from 0.019 to 0.239 nmol/mg and the 1 alpha-hydroxylase activity from 4.75 to 268 pmol/h per mg. Activity of the 24-hydroxylase in the crude solubilized extract was 6.3 pmol/h per mg, but was undetectable after partial purification by a p-chloroamphetamine-Sepharose column. However, further fractionation of this material by DEAE-Sepharose chromatography resulted in a further increase in 1 alpha-hydroxylase activity to 430 pmol/h per mg and detection of 24-hydroxylase in a separate fraction at a level of 53 pmol/h per mg. Production of 1,25-(OH)2-D3 was linear with time up to 2 h and was dependent upon ferredoxin concentration as well as cytochrome P-450 concentration in the range of 0-40 nM. In the presence of excess ferredoxin and adequate amounts of cytochrome P-450, 1,25-(OH)2-D3 production was also dependent upon substrate concentrations in the range of 0.25 to 2.5 microM yielding an estimated Km of 1 microM. In the presence of excess substrate and ferredoxin, the catalytic-centre activity of the enzyme was estimated to be 1 h-1.
pubmed:grant
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-14209971, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-205619, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-227393, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-2997195, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-3102023, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-318387, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-3702710, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-3718974, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-3838742, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-4074349, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-4151488, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-4310833, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-500724, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-50718, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6087030, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6600452, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6605973, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6684659, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6687381, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6807301, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-6890063, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-7011180, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-819434, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-861200, http://linkedlifedata.com/resource/pubmed/commentcorrection/2719667-942051
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0264-6021
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
259
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
561-8
pubmed:dateRevised
2009-11-18
pubmed:meshHeading
pubmed:year
1989
pubmed:articleTitle
Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases from vitamin D-replete pigs.
pubmed:affiliation
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.