Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells.
Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.
Ishizuka
Y
Y
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki.
Shoda
A
A
Yoshida
S
S
Kawamura
Y
Y
Haraguchi
K
K
Murakami
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000302:isolation & purification; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012083:Renin / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection
D002241:Carbohydrates; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012083:Renin