Abstract
A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.
Highlights
A 60-kDa protein homologous to phosphoinositidespecific phospholipase C - a was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW
We report the purification of eight types of protein from the rough endoplasmic reticulum (ER) of rat liver that are regarded as members of the phosphoinositide-specificphospholipase C-a family on the basis of their immunologicalcharacteristics and NHz-terminalamino acid sequences
The suspension was centrifuged for 10 min at 10,000 X g, and 0.5 p1 of the supernatant was subjected to two-dimensional electrophoresis
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No., Issue of July 25, pp. 15152-15159,1992 Printed in U.S.A. A 60-kDa protein homologous to phosphoinositidespecific phospholipase C - a was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. We report the purification of eight types of protein from the rough ER of rat liver that are regarded as members of the phosphoinositide-specificphospholipase C-a family on the basis of their immunologicalcharacteristics and NHz-terminalamino acid sequences. They did not show any phosphoinositide-specific phospholipase C activity, but showed proteolytic activity that was inhibited by p-chloromercuribenzoate (pCMB).
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