Tedesco T A, Mellman W J
J Clin Invest. 1969 Dec;48(12):2390-7. doi: 10.1172/JCI106205.
The specific activities of galactokinase and galactose-1-phosphate uridyltransferase were determined in peripheral blood leukocytes directly after separation from whole blood, and in cultured skin fibroblasts at various times during the subculture growth period. Growth curves were obtained for fibroblasts based on three different parameters: direct cell counts, total protein, and total deoxyribonucleic acid (DNA) content. At the time in culture when the specific activity of both enzymes was maximal and least variable, the ratio of transferase to galactokinase correlated well with the transferase genotypes of the original tissue donors. Leukocyte transferase: galactokinase ratios gave a similar distribution pattern. Whereas transferase activity in both fibroblasts and leukocytes was similar, galactokinase was approximately three times as active in fibroblasts as in leukocytes. All fibrobast cell strains tested had similar galactokinase activity regardless of transferase genotype.The kinetic properties of fibroblast galactokinase were examined. Galactose-1-phosphate inhibits galactokinase activity in both normal and galactosemic cell strains, whereas other glycolytic intermediates have no effect. There was no detectable transferase activity in eight galactosemic (Gt(G)/Gt(G)) cell strains when transferase activity was maximal in cell strains of other transferase genotypes. Inhibitors responsible for the absence of transferase activity could not be demonstrated. In addition, transferase activity in galactosemic cell lysates was not observed in cells during logarithmic growth; measurable uridine diphosphate galactose (UDPgal) pyrophosphorylase activity was found in human diploid fibroblast cultures, as well as significant levels of endogenous uridine triphosphate (UTP) in lysates of fibroblast cultures.
从全血中分离出来后，立即测定外周血白细胞中半乳糖激酶和1-磷酸半乳糖尿苷酰转移酶的比活性，并在传代培养生长期间的不同时间测定培养的皮肤成纤维细胞中的比活性。基于三个不同参数获得了成纤维细胞的生长曲线：直接细胞计数、总蛋白和总脱氧核糖核酸（DNA）含量。在培养过程中，当两种酶的比活性最高且变化最小时，转移酶与半乳糖激酶的比值与原始组织供体的转移酶基因型密切相关。白细胞转移酶：半乳糖激酶比值呈现出类似的分布模式。虽然成纤维细胞和白细胞中的转移酶活性相似，但成纤维细胞中的半乳糖激酶活性约为白细胞中的三倍。无论转移酶基因型如何，所有测试的成纤维细胞株都具有相似的半乳糖激酶活性。研究了成纤维细胞半乳糖激酶的动力学特性。1-磷酸半乳糖抑制正常细胞株和半乳糖血症细胞株中的半乳糖激酶活性，而其他糖酵解中间产物则没有影响。当其他转移酶基因型的细胞株中转移酶活性最高时，在八个半乳糖血症（Gt(G)/Gt(G)）细胞株中未检测到转移酶活性。未能证明导致转移酶活性缺失的抑制剂。此外，在对数生长期的细胞中未观察到半乳糖血症细胞裂解物中的转移酶活性；在人二倍体成纤维细胞培养物中发现了可测量的尿苷二磷酸半乳糖（UDPgal）焦磷酸化酶活性，以及成纤维细胞培养物裂解物中显著水平的内源性尿苷三磷酸（UTP）。
