Willick G E, Seligy V L
Eur J Biochem. 1985 Aug 15;151(1):89-96. doi: 10.1111/j.1432-1033.1985.tb09072.x.
The white-rot fungus, Schizophyllum commune, secretes a member of each of three classes of cellulases: a beta-glucosidase, an exoglucanase, and an endoglucanase. Antibodies were developed to members of each of these three enzyme classes. Secretion of these cellulases is induced when a mycelium is transferred from a glucose to cellulose medium. The maximum level of cellulase transcripts, as indicated by the ability to direct biosynthesis of these cellulases in the rabbit reticulocyte cell-free translation system, occurred when the rate of secretion was maximum. This implied that initial regulation, at least, of cellulase biosynthesis occurs at the transcriptional level. There were two distinct mRNA-directed products for each of the cellulases, with sizes estimated to be, for the beta-glucosidase, 95700 and 93800, for the exoglucanase, 59300 and 58200, and for the carboxymethylcellulase, 40600 and 39400. The secreted cellulases are largely glycosylated, as indicated by their binding to concanavalin A and their incorporation of D-[3H]mannose. The labelled protein was fractionated on concanavalin-A-agarose; about 70% of the label was bound. A small amount of each of the cellulases appeared in the unbound fraction; the remainder appeared in fractions eluted with 10 mM methyl glucoside or with 100 mM methyl glucoside plus 500 mM methyl mannoside. These results indicated each of the cellulases had an additional heterogeneity in glycosylation, with the most heavily glycosylated and highest molecular weight form eluting last from the concanavalin-A-agarose. Although tunicamycin (5 micrograms/ml) blocked glycosylation, there was still some secretion but at a reduced rate which was more pronounced for the beta-glucosidase than for the carboxymethylcellulase activity. The size of the tunicamycin-secreted product in each case was, within experimental error, equivalent to that of the mRNA-directed one.
一种β-葡萄糖苷酶、一种外切葡聚糖酶和一种内切葡聚糖酶。针对这三类酶的成员都制备了抗体。当菌丝体从葡萄糖培养基转移到纤维素培养基时，这些纤维素酶的分泌被诱导。如在兔网织红细胞无细胞翻译系统中指导这些纤维素酶生物合成的能力所表明的，纤维素酶转录本的最高水平出现在分泌速率最高时。这意味着至少纤维素酶生物合成的初始调节发生在转录水平。每种纤维素酶都有两种不同的mRNA指导产物，其大小估计为：β-葡萄糖苷酶为95700和93800，外切葡聚糖酶为59300和58200，羧甲基纤维素酶为40600和39400。分泌的纤维素酶大多被糖基化，这通过它们与伴刀豆球蛋白A的结合以及它们对D-[3H]甘露糖的掺入来表明。标记的蛋白质在伴刀豆球蛋白A-琼脂糖上进行分级分离；约70%的标记物被结合。每种纤维素酶的少量出现在未结合部分；其余部分出现在用10 mM甲基葡萄糖苷或100 mM甲基葡萄糖苷加500 mM甲基甘露糖苷洗脱的部分。这些结果表明每种纤维素酶在糖基化方面还有额外的异质性，糖基化程度最高和分子量最大的形式最后从伴刀豆球蛋白A-琼脂糖上洗脱下来。虽然衣霉素（5微克/毫升）阻断了糖基化，但仍有一些分泌，但速率降低，β-葡萄糖苷酶的这种情况比羧甲基纤维素酶活性更明显。在每种情况下，衣霉素分泌产物的大小在实验误差范围内与mRNA指导的产物相当。
