Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalyt ic (C) subunits were purified to homogeneity from soluble extract of Micros porum gypseum. Purified enzyme showed a final specific activity of 277.9 nm ol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa an d 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa s ubunit was found to be the regulatory (R) subunit of PKA holoenzyme as dete rmined by its immunoreactivity and the isoelectric point of this subunit wa s 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sep harose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, c ompatible with its tetrameric structure, consisting of two regulatory subun its (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein accepters in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had appar ent K-m values of 71 mu M and 25 mu M for ATP and kemptide, respectively. P hosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmale imide (NEM) which shows the involvement of sulfhydryl groups for the activi ty of PKA. PKA phosphorylated a number of endogenous proteins suggesting th e multifunctional role of cAMP dependent protein kinase in M. gypseum. Furt her work is under progress to identify the natural substrates of this enzym e through which it may regulate the enzymes involved in phospholipid metabo lism. (C) 2000 Elsevier Science B.V. All rights reserved.