Physical and Chemical Characterization of Pig Kidney Particulate Aminopeptidase

Wacker, Hans; Lehky, Pavel; Fischer, Edmond H.; Stein, Eric A.

doi:10.1002/hlca.19710540206

Cited by 42 publications

(12 citation statements)

References 35 publications

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“…The mobilities of the two smaller polypeptide chains differ slightly Vol. 159 from those observed by Wacker et al (1971) and this may well be attributable to slight differences in the carbohydrate content (Wacker, 1974). Although the -intact microvillus membrane contains polypeptide chains corresponding to apparent mol.wts.…”

Section: Dipeptidylpeptidase IVmentioning

confidence: 71%

“…Hence its location in the gel pattern has depended mainly on comparison with samples of the purified enzyme. Purified pig aminopeptidase M may be resolved by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate into three bands (Wacker et al, 1971 ;Wacker, 1974). The purification ofthis enzyme has so far required the use of proteolytic enzymes to release it from the membrane.…”

Section: Dipeptidylpeptidase IVmentioning

confidence: 99%

“…Several ofthekidneymicrovilluspeptidases have been purified and a number of these have now been shown to be glycoproteins. Aminopeptidase M (EC 3.4.11.2) contains about 20% carbohydrate (Wacker et al, 1971); neutral endopeptidase (EC 3.4.24.-) contains a similar proportion of carbohydrate (Kerr & Kenny, 1974b) as does y-glutamyltransferase (EC 2.3.2.2) (Szewczuk & Baranowski, 1963;Orlowski & Meister, 1965 Kenny, unpublished work) and kidney alkaline phosphatase (EC 3.1 .3. 1) has also been shown to be a glycoprotein (Binkley, 1961).…”

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confidence: 98%

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Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

Booth

Kenny

1976

Biochemical Journal

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The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as alpha-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

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Section: Dipeptidylpeptidase IVmentioning

confidence: 71%

Section: Dipeptidylpeptidase IVmentioning

confidence: 99%

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confidence: 98%

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Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

Booth

Kenny

1976

Biochemical Journal

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“…The Mg' +-ATPase and ouabain-sensitive Na+,K+-ATPase activities were determined using the coupled kinetic assay as modified by Scharschmidt et al (24). The activities of the second canalicular marker enzyme leucine aminopeptidase were measured according to Wacker et al (25). Glucagonstimulated adenylcyclase was chosen as a marker for the sinusoidal membrane domain and determined as previously described (26, 27).…”

Section: Methodsmentioning

confidence: 99%

Cryptic Na+, K+–Atpase Activity in Rat Liver Canalicular Plasma Membranes: Evidence for Its Basolateral Origin

et al. 1990

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Controversy exists concerning the localization of the enzyme Na+,K(+)-ATPase to canalicular membranes in hepatocytes. Most studies find enzyme activity only at the basolateral plasma membrane domain of the hepatocyte. However, Na+,K(+)-ATPase activity has been detected recently in a canalicular membrane fraction prepared by Mg++ precipitation, suggesting that differences in membrane domain fluidity account for these discrepancies. To reinvestigate this question, we used free-flow electrophoresis to further purify canalicular liver plasma membranes originally separated by sucrose density centrifugation. With this technique, canalicular membranes devoid of Na+,K(+)-ATPase activity by routine assay were separated into six subfractions. More than 80% of the activities of canalicular marker enzymes was recovered in two subfractions closest to the anode, which were totally devoid of Na+,K(+)-ATPase activity. However, Na+,K(+)-ATPase activity could now be detected in the four other fractions that contained only small amounts of canalicular marker enzymes. The basolateral marker enzyme, glucagon-stimulated adenyl cyclase, comigrated with this cryptic Na+,K(+)-ATPase activity. Furthermore, addition of 6 mumol/L [12-(2-methoxyethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate ], a membrane-fluidizing agent, to the original canalicular membrane preparation and to all subfractions did not stimulate or unmask latent Na+,K(+)-ATPase activity. Finally, when canalicular membranes isolated by Mg++ precipitation were subjected to free-flow electrophoresis, they could not be separated from the more positively charged Na+,K(+)-ATPase-containing fractions, probably because of alterations in surface charge. Together these findings suggest that Na+,K(+)-ATPase is a basolateral enzyme, that represents a small contaminant when present in canalicular liver plasma membranes and that methodological differences may account for previous discrepancies.

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“…Trakhanov. ~inopeptidase N activity in the vesicles was assayed spectrophotometrically on L-leucine pnitroanilide as substrate (at 405 nm) with a Gilford 2400-2 unit [17]. Protein concentration in the vesicles was measured by means of Coomassie brilliant blue G-250 [IS].…”

Section: Methodsmentioning

confidence: 99%

Role of intestinal brush border membrane aminopeptidase N in dipeptide transport

et al. 1984

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The kinetics of uptake of radioactive label from [U‐14C]Gly, L‐[4,5‐3H]Leu and the dipeptide [14C]Gly—L‐[4,5‐3H]Leu by the brush border membrane vesicles of porcine small intestine have been studied. The effect of aminopeptidase N inhibitors and leucine‐binding protein on accumulation rates has also been tested. Comparison of the kinetic parameters for uptake and hydrolysis of Gly—L‐Leu makes it possible to conclude that the dipeptide transfer includes two conjugated steps, viz., hydrolysis catalysed by aminopeptidase N and transport of the resultant free amino acids by a specific carrier.

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Physical and Chemical Characterization of Pig Kidney Particulate Aminopeptidase

Cited by 42 publications

References 35 publications

Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

Cryptic Na+, K+–Atpase Activity in Rat Liver Canalicular Plasma Membranes: Evidence for Its Basolateral Origin

Role of intestinal brush border membrane aminopeptidase N in dipeptide transport

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