Membrane protein phosphotyrosine phosphatase in rabbit kidney. Proteolysis activates the enzyme and generates soluble catalytic fragments.
Most protein phosphotyrosine phosphatases (PPT-phosphatases) have been recovered from the cytosol of various cell types and tissues. The present study explores the properties of PPT-phosphatases in rabbit kidney membranes prepared by centrifugation at 100,000 g. More of the total activity was recovered in membranes from fresh (45%) compared with frozen-and-thawed (36%) tissue. However, extracts of fresh tissue had only 15-30% as much total PPT-phosphatase activity. Up to 3-fold activation of cytosolic and membrane PPT-phosphatases occurred during preparation, an effect most evident when fresh tissue was homogenized in buffers containing multiple proteinase inhibitors. These inhibitors apparently block some, but not all, digestion of proteins that mask PPT-phosphatase activity. Incubation of membranes prepared from fresh tissue with added trypsin, papain or thermolysin in each case caused activation of PPT-phosphatase as well as generation of a soluble catalytic fragment. The fragment also was generated by the action of endogenous proteinases during repeated centrifugation and was isolated from these supernatants by DEAE-Sepharose, Zn2+-affinity and gel-filtration chromatography. The fragment had Mr approx. 33,000, had a neutral pH optimum, was inhibited by 50% by 100 microM-vanadate, and was insensitive to the alkaline-phosphatase inhibitors EDTA and levamisole. Although the chromatographic behaviour and lability of the fragment were distinct from those of the predominant cytosolic PPT-phosphatase, some cytosolic PPT-phosphatases exhibited properties consistent with the suggestion that they are fragments derived by proteolysis of PPT-phosphatases in membranes. Localization of PPT-phosphatases in plasma membranes would facilitate reaction with receptor/kinases in vivo.
Rotenberg
S A
SA
Division of Biology and Medicine, Brown University, Providence, RI 02912.
Brautigan
D L
DL
D000818:Animals; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007668:Kidney / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D008565:Membrane Proteins; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate; D014638:Vanadates; C028913:dodecyl sulfate; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D010450:Endopeptidases