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BACKGROUND: There is considerable evidence that calcium/calmodulin-dependent protein kinase II (CaM kinase II) plays a key role in insulin secretion and the enzyme provides a candidate gene for Type 2 diabetes. Since several isoforms of the enzyme exist, it is essential to define which are expressed by the beta-cell. METHODS: A human islet cDNA library in lambdaZAPII was screened with a probe for the 5'-end of human gamma CaM kinase II. Since this region is very homologous between the different isoforms, it is expected that isoforms other than gamma would be detected. From each of the six positive clones obtained, DNA was prepared and subjected to PCR using primers spanning the variable region in which the main variability of CaM kinase II isoforms resides. PCR products were purified and sequenced in both directions. The beta-cell line MIN6 was screened for CaM kinase II delta by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Western blotting. RESULTS: The sequences of five of the human islet PCR products indicated that the clones corresponded to the gamma(B) isoform whose expression in human islets we have previously documented. The other PCR product, however, gave a sequence containing the variable domains II and VII characteristic of CaM kinase II delta. This sequence and the absence of other domains in this region identified the clone as CaM kinase II delta(C). The expression of CaM kinase II delta in MIN6 beta-cells was confirmed by RT-PCR and by Western blotting. CONCLUSIONS: Human islets of Langerhans express the delta(C) isoform of CaM kinase II.

Original publication

DOI

10.1002/(sici)1520-7560(199907/08)15:4<243::aid-dmrr40>3.0.co;2-3

Type

Journal article

Journal

Diabetes Metab Res Rev

Publication Date

07/1999

Volume

15

Pages

243 - 246

Keywords

Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Gene Expression, Gene Library, Humans, Islets of Langerhans, Isoenzymes, Mice, Molecular Sequence Data, Reverse Transcriptase Polymerase Chain Reaction