PubMed · 5377384
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Y Yamamura. 1969. [Liver function tests and enzymes].. https://pubmed.ncbi.nlm.nih.gov/5377384/
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Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is characterized by varying degrees of developmental delay, motor and gait abnormalities, seizures, and thrombosis. Biochemical abnormalities include homocystinuria and hyperhomocysteinemia. Clinical severity correlates with MTHFR activity in cultured fibroblasts; activity can also be assayed in cultured amniocytes and chorionic villus cells (CVC). Forty-four private mutations have been identified, limiting the use of direct mutation detection for prenatal diagnosis. However, intragenic polymorphisms have been identified, making prenatal diagnosis by linkage analysis a possible option, even without knowledge of deleterious mutations. Prenatal diagnosis for severe MTHFR deficiency has been available by biochemical methodologies, but molecular genetic approaches have not yet been reported. We performed prenatal diagnosis for severe MTHFR deficiency in 11 at-risk pregnancies in seven families. A combined approach of linkage analysis and enzymatic assays was used in six pregnancies; linkage analysis alone was performed in one pregnancy. Linkage analysis for the 677C > T or 1298A > C polymorphisms predicted that all seven fetuses were unaffected. For six of these seven fetuses, enzymatic activities were also measured and demonstrated concordant results. Of the 10 pregnancies in which enzymatic assays were performed, activities in cultured amniocytes predicted six unaffected fetuses (1.4-7.1 nmol CHO/mg prot/h (U)) and one affected fetus (0.24 U [control 3.1-9.6 U]). Three pregnancies assessed via CVCs demonstrated two unaffected fetuses (3.6 and 7.7 U) and 1 affected fetus (0 U [control 4.5-7.8 U]). These values were compared to those of the probands (range = 0.02-0.7 U (control 2.4-11.7 U)) in cultured fibroblasts. Our findings suggest that linkage analysis for severe MTHFR deficiency can be a practical approach for prenatal diagnosis.
We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N-acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn(2+) concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes.
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