title: Purification and enzymatic characterization of recombinant prohormone convertase 2: stabilization of activity by 21 kDa 7B2.
abstract: Although previous efforts to produce significant quantities of purified prohormone convertase 2 from either recombinant or natural sources have been unsuccessful, our recent finding that the neuroendocrine polypeptide 7B2 is necessary for the biosynthesis of enzymatically active prohormone convertase 2 (PC2) has enabled us to obtain active recombinant enzyme from the conditioned medium of PC2-producing CHO cells supertransfected with cDNA coding for 21 kDa 7B2. The recombinant enzyme was purified to apparent homogeneity, with a 40% recovery, in milligram quantities. Two protein bands of Mrs 71 and 75 kDa were observed after SDS-PAGE followed by either Coomassie staining or Western blotting with PC2 antiserum. Spontaneous conversion of the 71- and 75-kDa species to the 66-kDa form occurred during incubation at pH 5.0; the degree of conversion correlated with a dramatic increase in activity. Kms of 124 and 131 microM and Kcats of 0.49 and 0.81 s(-1) were obtained for the substrates Cbz-Arg-Ser-Lys-Arg-AMC and Pyr-Arg-Thr-Lys-Arg-AMC, respectively. The pH optimum was 5.0, and the enzyme was inhibited by h7B2(155-185') p-CMS, and EDTA but not by other inhibitors tested. Interestingly, 21 kDa 7B2 was observed to copurify with the enzyme in a molar ratio of about 1:100 (7B2:PC2). Prior addition of recombinant 21 kDa 7B2 to activated 66 kDa PC2 provided significant protection against thermal denaturation. When coassociated 7B2 was mostly removed from activated PC2 through gel filtration, subsequent addition of recombinant 7B2 exerted a significant stabilizing effect on enzyme activity. Millimolar Ca2+ and pHs between 5 and 6 were required to observe this effect. Since these conditions resemble those thought to occur within secretory granules, and since 21 kDa 7B2 represents a stored secretory granule protein, our data suggest a physiological role for 21 kDa 7B2 in the stabilization of PC2 activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Lamango
authors.forename: X
authors.initials: X
authors.lastname: Zhu
authors.forename: I
authors.initials: I
authors.lastname: Lindberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004745:Enkephalins / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051859:Neuroendocrine Secretory Protein 7B2; D009842:Oligopeptides / Q000737:chemistry; D010907:Pituitary Hormones / Q000378:metabolism*; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
chemical_list: D017931:DNA Primers; D004745:Enkephalins; D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D009842:Oligopeptides; D010907:Pituitary Hormones; D011498:Protein Precursors; D011994:Recombinant Proteins; C497678:SCG5 protein, human; C497680:Scg5 protein, rat; C029992:proenkephalin; D013381:Subtilisins; D045706:Proprotein Convertase 2