(1) The Mg(II)-ATP-dependent protein phosphate is composed of two types of subunits. The catalytic subunit C migrates at 38K Da during SDS-gel electrophoresis and the modulator subunit M at 31K Da. This enzyme is inactive as isolated. It is activated by protein kinase FA which is the same as glycogen synthase kinase-3, and MgATP. Quantitation of this enzyme in crude extracts shows that it is present in high concentration (about 50 nM). In addition, the maximal specific activity of the purified enzyme is enzyme concentration dependent. Further study shows that the enzyme is composed of two modulator and one catalytic subunit as evidenced by the data obtained from (i) gel filtration and sucrose gradient centrifugation experiments, (ii) densitometric scans of silver stained SDS-polyacrylamide gels, (iii) calculations based on amino acid analysis of the separated subunits; (iv) deconvolution analysis of the UV spectra of the denatured enzyme complex, and (v) steady-state kinetic analysis of the enzyme inhibition by added modulator subunit. One modulator binds very tightly to the catalytic subunit, while the other is dissociable and functions as a competitive inhibitor for the substrate. Thus, it provides a mechanism for protecting the phosphoproteins present in low concentrations. (2) Development of a model for interactions between transmembrane proteins and the membrane potential. The theory can be used to provide a reasonable interpretation for energy transduction and for explaining how two a priori independent reactions can be coupled by an enzyme. (3) Rapid intracellular changes in both calcium and pH concentration in sperm of sea urchin Strongylocentratus purpuratus caused by speract and by egg coat were measured.
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