Structural distinction between soluble and particulate protein kinase C species.
A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca2+/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more alpha-helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200-250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.
Lester
D S
DS
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Orr
N
N
Brumfeld
V
V
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D004161:Diphenylhexatriene; D005583:Fourier Analysis; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009282:Naphthalenesulfonates; D010770:Phosphotransferases / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods; D013055:Spectrophotometry, Infrared / Q000379:methods; D014364:Tryptophan
D008565:Membrane Proteins; D009282:Naphthalenesulfonates; D004161:Diphenylhexatriene; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014364:Tryptophan; D010770:Phosphotransferases; D011493:Protein Kinase C