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Looking for evidence that flavin containing oxidase involves pyrroloquinoline quinone

寻找含黄素氧化酶涉及吡咯喹啉醌的证据

基本信息

批准号：
61560124
负责人：
ADACHI Osao
金额：
$ 0.96万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Several flavin containing oxidases, such as xanthine oxidase, D-amino acid oxidase, glucose oxidase, and other enzymes which have been assumed to contain flavin, eg. uricase or ascorbate oxidase, were hydrolyzed by proteolysis and/or acid hydrolysis. The resulting hydrolysates were andlyzed to see whether exist any signal showing the presence of pyrroloquinoline quinone (PQQ) or PQQ-chromophores. After fractionation with Sephadex G-10 column, each fraction was examined of its activity as the prosthetic group for apo-glucose dehydrogenase (EC 1.1.99.17). From the chromatogram of some reference proteins in which PQQ is never involved, such as lysozyme and NAD-dependent alcohol dehydrogenase of several recrystallized preparations, showed no PQQ activity. On the other hand, of the examine candidates, the hydrolysate from uricase, xanthine oxidase, ascorbate oxidase potently showed the presence of PQQ in their molecule. These hydrolysate were also available as the growth stimulating substance for some microorganisms to reduce the lag phase in their growth. Spectroscopic data indicated the possibility of the existence of PQQ or PQQ-related compound in individual enzyme molecule.Alternative model experiment was carried out with PQQ and bovine serum albumin. When PQQ was mixed with bovine serum albumin at neutral pH, a spontaneous binding of PQQ to serum albumin was observed. The resulting PQQ-BSA was fairly stable for chromatographic treatments and exhibited absorption spectrum having a peak at 336 nm. PQQ was convinced to be asociated via ipsilon amino group of lysyl residue of BSA via a Schiff base.

几种含有黄素的氧化酶，如黄嘌呤氧化酶、D-氨基酸氧化酶、葡萄糖氧化酶和其他被认为含有黄素的酶，如葡萄糖氧化酶。尿酸酶或抗坏血酸氧化酶通过蛋白水解和/或酸水解来水解。分析所得水解产物以观察是否存在任何显示吡咯并喹啉醌（PQQ）或PQQ-发色团存在的信号。在用Sephadex G-10柱分级分离后，检测每个级分作为脱辅基葡萄糖脱氢酶的活性（EC 1.1.99.17）。从一些参考蛋白质的色谱图中，其中PQQ从来没有参与，如溶菌酶和NAD依赖性醇脱氢酶的几个重结晶制剂，显示没有PQQ活性。另一方面，在待测的候选物中，尿酸酶、黄嘌呤氧化酶、抗坏血酸氧化酶的水解产物有力地显示了PQQ在其分子中的存在。这些水解物也可用作某些微生物的生长刺激物质，以减少其生长的滞后期。光谱数据表明，在单个酶分子中可能存在PQQ或PQQ相关化合物。当PQQ与牛血清白蛋白在中性pH下混合时，观察到PQQ与血清白蛋白的自发结合。所得PQQ-BSA对于色谱处理相当稳定，并显示出在336 nm处具有峰的吸收光谱。PQQ通过Schiff碱与BSA赖氨酰残基的异丙氨基缔合。