Linked Life Data

3091605

Source:http://linkedlifedata.com/resource/pubmed/id/3091605

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
27
pubmed:dateCreated
1986-10-23
pubmed:abstractText
Procedures have been developed for the purification of a nearly homogeneous, highly active phosphate transport system from rat liver mitochondria in either a two-subunit (alpha, beta) or a single subunit (beta) form. Significantly, both forms display a similar high magnitude N-ethylmaleimide (NEM)-sensitive Pi/Pi exchange activity upon incorporation into phospholipid vesicles. The transport system is extracted from hypotonically shocked mitoplasts with Triton X-114 and purified in the presence of cardiolipin by sequential chromatography on hydroxylapatite, DEAE-Sepharose CL-6B, and Affi-Gel 501. Depending on the conditions used to elute the transporter from Affi-Gel 501, preparations are obtained which, when analyzed by high resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, consist of either a single 33-kDa protein (beta) or a 33-kDa (beta) plus a 35-kDa (alpha) component. In preparations yielding the latter result, both bands display a nearly equivalent Coomassie staining intensity. Furthermore, after alkylation with NEM, the two protein bands co-migrate. Fluorography indicates that the coalesced band contains [3H]NEM. Upon reconstitution of the purified Pi carrier into liposomes, direct measurement of both the initial transport rate and the amount of protein that actually incorporates into the phospholipid vesicles yields a specific transport activity of 22.6 mumol/min/mg of protein. The exchange is characterized by a first order rate constant of 0.85 min-1, a t1/2 of 49 s, and is inhibited by sulfhydryl reagents (i.e., NEM, p-chloromercuribenzoate, and mersalyl). It is also substantially inhibited by diethyl pyrocarbonate, N-acetylimidazole, phenylglyoxal, and 5-dimethylaminoaphthalene-1-sulfonyl chloride. In addition to providing a simple, rapid method for preparing the NEM-sensitive phosphate carrier in nearly homogeneous form, these studies provide new information about the catalytically active species of the carrier, its kinetic properties, and its inhibitor sensitivities.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
25
pubmed:volume
261
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12767-73
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1986
pubmed:articleTitle
Purification and characterization of the reconstitutively active phosphate transporter from rat liver mitochondria.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.