Abstract
Creatine kinase enzymes are present in tissues such as muscle and brain to interconvert creatine phosphate and ADP, thus providing a system to interconnect energy production and utilization (Bessman, S. P., and Carpenter, C. L. (1985) Annu. Rev. Biochem. 54, 831-862). Creatine kinase isoenzymes in kidney have received little attention since kidney contains relatively low creatine kinase activity compared with muscle and brain and because there is disagreement regarding the identity of the specific isoforms expressed in kidney. Using a combination of chromatographic and immunological techniques, we have identified two isoforms of creatine kinase in rat kidney supernatants, B creatine kinase, and the non-sarcomeric form of the mitochondrial creatine kinase, which represent 82 and 15%, respectively, of the total creatine kinase activity in this tissue. The identity of the non-muscle form of the mitochondrial creatine kinase was confirmed by N-terminal sequence analysis and compared with recently published cDNA sequences (Haas, R. C., and Strauss, A. W. (1990) J. Biol. Chem. 265, 6921-6927). We prepared multiple antisera specific for each isoform using synthetic peptide immunogens based upon nonhomologous regions from the primary sequence of each creatine kinase isoform. Immunocytochemical results demonstrate that both creatine kinase isoforms are colocalized in the inner stripe of the outer medulla in tubules of the distal nephron. A similar distribution of creatine kinase isoforms was obtained when different layers of the renal cortex and medulla were examined for creatine kinase activity and isozyme content using nondenaturing electrophoresis. In general, the distribution of creatine kinase enzymes in kidney corresponds to the regions of greatest ATP utilization, oxygen consumption, and sodium transport. These results suggest a role for creatine kinase enzymes in the coupling of ion transport and oxidative phosphorylation in the distal nephron of the mammalian kidney.
Highlights
11 2 3 4 5 1 6 7 8 9110 11 12 13114 15 16 17 181 creatine kinasesequence (168 and253; see Table I) recognize antisera 110; lanes 7, 12, and 16, antisera 34; lane 3, antisera 168; lanes 4, 10, and 15, antisera 253; lanes 2, 9,13, and 14, antisera 165
The results of this study demonstrate the presence of two creatine kinase isoforms, BB and non-muscle mitochondrial creatine kinase,in the distal nephronof the ratkidney
The ratio of the BB creatine kinase activity to the mitochondrial creatine kinase activity can be estimated from the DEAEcolumn profile (Fig. 1).The mitochondrialcreatinekinaseactivityconstitutes approxii "
Summary
Mercaptoethanol (5 mM) and centrifuged (12,000 X g, 10 rnin.). Cytosolic fractions from rat kidneywere analyzed for creatine kinase isoenzymes using nondenaturing conditions. Supernatants prepared from these regions were analyzed by Creatine kinase activity was measured by spectrophotometry ac- nondenaturing electrophoresis as shown in lanes 1-4. Western Blot Analysis-Tissue extracts (75-100 pgs) and purified proteins (1-3 pgs) were resolved on gradient (7.5-20%) SDS'-polyacrylamide gels and subsequently transferred to nitrocellulose membranes.Themembranes were incubated for 60 min in ablocking buffer consistingof 50 mM Tris-HCI, 0.5 M NaCI, 4% powdered milk, and 1% bovine serum albumin. The specific creatine kinase activity is 0.886 IU/mg of protein in the cortex and 0.884 IU/mg of protein in the outer stripe. Creatine kinase activity is about 6-fold greater (specific activity = 4.88 IU/mg of protein). The first peak fromtheDEAE profile (fractions 1-19; Fig. 2B, lune I ) contains a doubletwhich comigrates with mitochondrial creatine kinase purified from rat brain (Fig. 2R, lune 3 ).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
