Purification And Characterization Of Protein Phosphatase C From Rabbit Muscle
Date of Award
Doctor of Philosophy (Ph.D.)
Biochemistry and Molecular Biology
The process of phosphorylation-dephosphorylation of the enzymes of glycogen metabolism is known to be an important regulatory mechanism. Although the enzymology of the protein kinases has been well characterized, much less is known about the protein phosphatases, which catalyze the reverse process of dephosphorylation. This study describes the purification and characterization of protein phosphatase C generated by ethanol treatment of rabbit muscle extracts. The use of a linear salt gradient on a long DEAE-Sepharose column led to the separation and isolation of two forms of low molecular weight protein phosphatase. Peak I, a high specific activity phosphorylase phosphatase (11,300 Units/mg), and Peak II, a low specific activity phosphorylase phosphatase (555 Units/mg) were named as such because of their respective orders of elution from an initial DEAE-Sepharose column. The relatively simple purification scheme developed made possible the isolation of both Peak I and Peak II to apparent homogeneity (on SDS-gel electrophoresis). Gel filtration on Sephadex G-75 revealed that both forms of protein phosphatase eluted at the same position with a M(,r) = 35,000 daltons. On sodium-dodecyl sulfate gel electrophoresis Peak I (M(,r) ca. 32,000) was found as the smaller of the two and Peak II (the slightly larger form) had a molecular weight M(,r) = 33,500.Non-denaturing polyacrylamide electrophoresis and sucrose density gradient ultracentrifugation of purified Peak I and Peak II showed significant distinctions in the mobilities of each form. Peak I had a R(,m) of 0.48, and S(,20)W values of 3.3-3.4; Peak II migrated with an R(,m) of 0.68, and had S(,20)W values of 3.5-3.6. Characteristic levels of several intrinsic phosphatase activities (recoverable) present in Peak I or Peak II were revealed by these non-denaturing methods. In addition to high phosphorylase phosphatase activity Peak I co-migrates with histone phosphatase, Mn('++) inhibited phosphorylase kinase phosphatase, and glycogen synthase phosphatase activities.The amino acid compositions, nature of fragments formed on CNBr cleavages, and partial proteolysis of SDS-treated enzyme all strongly indicate that Peak I and Peak II have distinct amino acid sequences. Previously reported and even recent studies using low molecular weight "purified" protein phosphatase may in fact have used preparations consisting of a mixture of these different forms. Results obtained with protease inhibitors negates the involvement of proteases in the generation of M(,r) 35,000 protein phosphatase during ethanol treatment. The implications derived from the isolation of these two forms of protein phosphatase C is that in rabbit muscle several (M(,r) 260,000) holoenzyme forms exist consisting of (M(,r) 35,000) catalytic subunits that have different physical and biochemical properties.
Silberman, Steven Randall, "Purification And Characterization Of Protein Phosphatase C From Rabbit Muscle" (1981). Dissertations from ProQuest. 1249.