Hutton J C, Davidson H W, Peshavaria M
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Biochem J. 1987 Jun 1;244(2):457-64. doi: 10.1042/bj2440457.
The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.
在可移植胰岛素瘤组织中，研究了负责从其100 kDa嗜铬粒蛋白A样前体产生20 kDa蛋白β颗粒蛋白的酶活性的性质和亚细胞定位。经裂解的胰岛素分泌颗粒将[³⁵S]甲硫氨酸标记的前体转化为β颗粒蛋白和一种或多种47 kDa的蛋白质，通过60 - 65 kDa范围内的中间体进行转化。富含溶酶体的组分也能处理前体，但不会转化为体内发现的肽；其他组分，包括富含高尔基体的组分，没有活性。颗粒对前体的转化是定量的，且产物稳定。抑制剂研究表明，加工过程是通过在由成对碱性氨基酸标记的位点进行初始内切蛋白水解切割，然后由羧肽酶H去除这些碱性氨基酸。内切肽酶活性似乎是一种新型金属酶，最适pH值明显呈酸性（4.8 - 5）。它被丙氨酰-L-赖氨酰-L-精氨酰氯甲烷抑制（K₀.₅ = 1.3 μM），但被由单个或不成对碱性氨基酸残基定义的加工位点的抑制剂类似物抑制的程度要小得多，例如丙氨酰-L-正亮氨酰-L-精氨酰氯甲烷（K₀.₅大于100 μM）、亮抑酶肽（K₀.₅ = 150 μM）和抗蛋白酶（K₀.₅ = 40 μM）。对氯汞苯甲酸（K₀.₅ = 13 μM）、Hg²⁺（K₀.₅ = 16 μM）、Zn²⁺（K₀.₅ = 0.8 mM）和钒酸盐（K₀.₅ = 7 μM）也能消除活性，各种阴离子（SCN⁻＞I⁻＞Cl⁻＞SO₄²⁻）也是如此。丝氨酸、巯基和酸性内切肽酶的基团特异性抑制剂没有作用。EDTA和CDTA（1,2 - 环己二胺四乙酸）能消除内切蛋白水解活性，但1,10 - 菲咯啉不能。几种二价阳离子在EDTA或CDTA抑制后可以恢复活性，包括Ca²⁺、Zn²⁺、Mn²⁺和Sr²⁺；然而，具有生理重要性的离子似乎是Ca²⁺（K₀.₅ = 8 μM）。颗粒内切肽酶的性质及其亚细胞定位表明，它在胰腺β细胞中处理嗜铬粒蛋白A方面具有重要作用。
