Sachiko Nakagawa

Human hexosaminidase isozymes: chromatographic separation as an aid to heterozygote identification.

The correct identification of Tay-Sachs heterozygotes requires a reliable procedure for separation and quantiation of the hexosaminidase isozymes. The most commonly employed method involves thermal inactivation of the heat labile hexosaminidase A assay of residual enzyme activity. This procedure, however, consistently yields a significantly lower absolute and relative activity of hexosaminidase A and a higher activity of the thermostable components (B and I) in comparison with the results obtained by DEAE-cellulose chromatography. DEAE-cellulose chromatographic separation of the hexosaminidase isozymes in serum following thermal inactivation reveals the presence of relative and absolute increase in the activity of the B and I components in addition to loss of the heat-labile A isozyme. Because the conversion of hexosaminidase A into thermostable forms by heating may vary according to the conditions employed, the thermal inactivation procedure may lead to ambiguity in heterozygote identification. This difficulty can be minimized by fractionation of the hexosaminidase isozymes by DEAE-cellulose chromatography followed by assay of the individual components. In addition to the Tay-Sachs carrier state, other conditions can alter the distribution of the hexosaminidase isozymes in tissues and body fluids. For example in serum of patients with juvenile diabetes mellitus there is a characteristic elevation of hexosaminidase B and less consistently, of hexosaminidase A. Since the activity of hexosaminidase A in serum of diabetics fractionated by ion exchange chromatography is at least as high as the activity in serum of healthy non-carriers, patients with diabetes can be easily differentiated from Tay-Sachs heterozygotes. Similarly, the distribution of the hexosaminidase isozymes in serum is altered during pregnancy, where there is usually a significant rise in hexosaminidase A and I (P). However, during pregnancy activities of hexosaminidase A and I in serum of obligate Tay-Sachs carriers are only 50% of the values observed in non-carriers at comparable gestational periods. Since the absolute activities of hexosaminidase A in serum of pregnant carriers obtained by ion exchange chromatography do not overlap with the activities in serum of non-carrier pregnant women at comparable gestational periods, this method has obvious advantages for identification of pregnancies where the fetus may be at risk for Tay-Sachs disease.

Altered Lysosomal Glycohydrolase Activities in Juvenile Diabetes Mellitus

Studies have been carried out on activities of lysosomal β-N-acetylhexosaminidase (hex), β-galactosidase (β-gal), α-glucosidase (α-glu), and acid phosphatase (AP) in serum and urine from patients with juvenile diabetes and matched controls. There is a large increase in blood and urinary hex activity (the former presenting three distinct patterns of abnormality), a moderate increase in urinary β-gal, and a small increase in urinary α-glu activity, but no elevation of blood or urinary AP in the diabetics. Urinary α-glu activity in the diabetics shows striking inhibition by glucose, and this may reflect a similar phenomenon in vivo. Although glycohydrolase activities are elevated in patients with no detectable microangiopathy, more striking changes may be observed in patients with severe small-vessel disease. These alterations may be associated with increased glycoprotein catabolism in the diabetic, an area in need of further studies in the human and experimental diabetic animal.

Human hexosaminidase isozymes. Assay of platelet activity for heterozygote identification during pregnancy.

Identification of carriers of the Tay-Sachs gene during pregnancy is difficult because of the increase in serum of a heat stable hexosaminidase isozyme I (or P) as well as changes in the relative and absolute activities of the various molecular forms of the enzyme with advancing pregnancy. In contrast, isolation of blood platelets followed by ion exchange chromatographic separation and assay of the hexosaminidase isozymes in platelet extracts by an automated method provides a sensitive and reliable method for heterozygote identification during pregnancy. This method appears superior to procedures involving thermal inactivation of extracts of peripheral blood leukocytes because of significant differences in the content of the hexosaminidase isozymes in granulocytes, lymphocytes and other cell types, as well as variations in the proportion of these cell types in samples of peripheral blood. It also alleviates the problem inherent in any method involving thermal inactivation of hexosaminidase A by avoiding possible interconversion of the various molecular forms of the enzyme associated with heating.

Studies on the activities and properties of lysosomal hydrolases in fractionated populations of human peripheral blood cells

Studies have been carried out on the activities and properties of the isozymes of alpha-mannosidase, alpha-glucosidase and beta-glucosidase in granulocytes, monocytes, lymphocytes and platelts from peripheral blood of heatlhy adult donors. The findings reveal the differences in activities as well as a characteristic distribution of the different molecular forms of these lysosomal hydrolases in specific cell types. Therefore, the results obtained with unfractionated total leukocyte smples from different subjects may vary according to the distribution of cell types in the circulation. Granulocytes and monocytes show only the acid alpha-mannosidase activity whereas lymphocytes and platelets show both acid and neutral activities. The specific activity of acid alpha-mannosidase in granulocytes and monocytes is higher than in lymphocytes and platelets. By DEAE-cellulose chromatography, the acid alpha-mannosidase in granulocyte and monocyte extracts elutes as two peaks, but only one peak is seen in lymphocytes. All cell types show both acid and neutral alpha-glucosidase activities. The specific activities of both isozymes are higher in granulocytes and monocytes than in lymphocytes and platelets. Monocytes show a higher acid than neutral activity. All other cell types show a higher neutral activity. Beta-Glucosidase in all cell types is mainly membrane-bound and it can be released by Triton X-100 and sodium taurocholate. Taurocholate also stimulates the beta-glucosidase activity of granulocytes, monocytes and lymphocytes whereas it inhibits the activity of this enzyme in platelets. These results indicate that variations in the total number of leukocytes and in the relative proportion of the various cell types in health and disease may yield inconsistent or unreliable values for enzyme activity in the diagnosis of lysosomal storage disease and in carrier detection.

Heterozygote detection of Type I Gaucher disease using blood platelets

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of beta-glucosidase and 4-methylumbelliferyl-beta-D-glucoside as substrate. Platelet lysates have at least two identifiable beta-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the beta-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of beta-hexosaminidase to beta-glucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of beta-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of beta-glucosidase at pH 5.6 in the presence of sodium taurocholate with the ratio of beta-hexosaminidase to beta-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.

A comparative study of the properties of rat and human N-acetyl-β--hexosaminidase

Abstract 1. 1. There are four hexosaminidase isozymes in rat serum. One is thermostable and the remaining three are thermolabile. Human serum contains three isozymes; two are thermostable and one is thermolabile. In general, the hexosaminidase isozymes in rat serum are more thermolabile than those in human serum. In contrast, the kinetic properties of the hexosaminidase isozymes in serum from the two species are similar. 2. 2. Rat liver and human placenta contain two hexosaminidase isozymes one of which is thermolabile and the other thermostable. These tissue isozymes from the two species have similar physical and kinetic properties.

Decreased activity of a soluble DNA-dependent RNA polymerase from thymus of rats injected with a thymolytic steroid

Abstract The Mg 2+ - and Mn 2+ - catalyzed DNA-dependent RNA polymerase activity of rat thymic nuclei has been solubilized by three procedures. The soluble preparation required addition of DNA for activity, indicating that endogenous DNA had been separated from the enzyme. Enzymatic activity solubilized from thymocytes of rats injected with either cortisol or fluocinolone acetonide 3 hr prior to sacrifice was significantly lower than that from cells of control rats. This decreased DNA-dependent RNA polymerase activity is due, at least in part, to an alteration in the enzymatic component of the DNA-RNA polymerase complex.

Elevation of serum β-hexosaminidase and α-d-mannosidase in type 2 gaucher disease: a clinical and biochemical study

We report a case of a black infant who died at 9 months of age with clinical and pathological findings consistent with the acute neuronopathic form of Gaucher disease (Type 2). Analysis of peripheral blood platelets obtained from this child demonstrated very low levels of β-glucosidase activity. β-hexosaminidase (HEX) activity in the serum, however, was 30 times greater than the level in control sera and 15 times greater than the level observed in individuals affected with the chronic form of Gaucher disease (Type 1). Similarly, α-d-mannosidase (MANN) activity in the probands serum was significantly elevated when compared with controls, and chronic Gaucher disease patients. We postulate that the cause of the elevation of these lysosomal enzymes is similar to the cause of elevation in Type 1 individuals but that patients with Type 2 Gaucher disease have a more serious cellular defect.

Human hexosaminidase isozymes. IV. Effects of oral contraceptive steroids on serum hexosaminidase activity.

Data from a mass screening program for identification of persons who are heterozygous for Tay-Sachs disease have been analyzed for the effects of oral contraceptive steroids on the activity in serum of hexosaminidase (hex) and the various hex isozymes. Women using oral contraceptives show a significantly higher total serum hex activity, reflecting mainly an increase in the heat-stable hex isozyme (hex I), and a smaller increase in the heat-labile hex A than do women using no medications. The changes in women using oral contraceptives are qualitatively similar to those observed during pregnancy. The distribution of responses to oral contraceptives is unimodal, and some of the variations may be related to differences in amount of steroid ingested. In addition, underlying genetic variation may contribute to the observed differences in enzyme activities.