B Hoffmann, C Seib, A Höer, D Höer, E Oberdisse, W Rosenthal, G Schultz
{"title":"Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.","authors":"B Hoffmann, C Seib, A Höer, D Höer, E Oberdisse, W Rosenthal, G Schultz","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.</p>","PeriodicalId":8948,"journal":{"name":"Biomedica biochimica acta","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1991-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biomedica biochimica acta","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.