CHARACTERIZATION OF HUMAN ACID ALPHA-GLUCOSIDASE

The goal of this study was to investigate the properties of human acid (alpha)-glucosidase with respect to: (i) the molecular heterogeneity of the enzyme and (ii) the synthesis, post-translational modification, and transport of acid (alpha)-glucosidase in human fibroblasts.^ The initial phase of these investigations involved the purification of acid (alpha)-glucosidase from the human liver. Human hepatic acid (alpha)-glucosidase was characterized by isoelectric focusing and native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four distinct charge forms of hepatic acid (alpha)-glucosidase were separated by chromatofocusing and characterized individually. Charge heterogeneity was demonstrated to result from differences in the polypeptide components of each charge form.^ The second aspect of this research focused on the biosynthesis and the intracellular processing and transport of acid (alpha)-glucosidase in human fibroblasts. These experiments were accomplished by immune precipitation of the biosynthetic intermediates of acid (alpha)-glucosidase from radioactively labeled fibroblasts with polyclonal and monoclonal antibodies raised against human hepatic acid (alpha)-glucosidase. The immune precipitated biosynthetic forms of acid (alpha)-glucosidase were analyzed by SDS-PAGE and autoradiography. The pulse-chase experiments demonstrated the existence of several transient, high molecular weight precursors of acid (alpha)-glucosidase. These precursors were demonstrated to be intermediates of acid (alpha)-glucosidase at different stages of transport and processing in the Golgi apparatus. Other experiments were performed to examine the role of co-translational glycosylation of acid (alpha)-glucosidase in the transport and processing of precursors of this enzyme.^ A specific immunological assay for detecting acid (alpha)-glucosidase was developed using the monoclonal antibodies described above. This method was modified to increase the sensitivity of the assay by utilization of the biotin-avidin amplification system. This method was demonstrated to be more sensitive for detecting human acid (alpha)-glucosidase than the currently used biochemical assay for acid (alpha)-glucosidase activity. It was also demonstrated that the biotin-avidin immunoassay could discriminate between normal and acid (alpha)-glucosidase deficient fibroblasts, thus providing an alternative approach to detecting this inborn error in metabolism. (Abstract shortened with permission of author.) ^

An evaluation of the Cypress Creek watershed for inorganic chemical load as it flows into Lake Houston

Background. The Cypress Creek is one of the main tributaries of Lake Houston, which provides drinking water to 21.4 million customers. Furthermore, the watershed is being utilized for contact and non-contact recreation, such as canoeing, swimming, hiking trail, and picnics. Water along the creek is impacted by numerous wastewater outfalls from both point and non-point sources. As the creek flows into Lake Houston, it carries both organic and inorganic contaminants that may affect the drinking water quality of this important water source reservoir. Objective. This study was carried out to evaluate the inorganic chemical load of the water in Cypress Creek along its entire length, from the headwaters in Waller County and up to the drainage into Lake Houston. The purpose was to determine whether there are hazardous concentrations of metals in the water and what would be the likely sources. Method. Samples were collected at 29 sites along the creek and analyzed for 29 metals, 17 of which were on the Environmental Protection Agency priority pollution list. Public access sites primarily at bridges were used for sample collection. Samples were transported on ice to the University Of Texas School Of Public Health laboratory, spiked with 2 ml HNO3 kept overnight in the refrigerator, and the following day transported to the EPA laboratory for analysis. Analysis was done by EPA Method 200.7-ICP, Method 200.8ICP/MS and Method 245.1-CVAAS. Results. Metals were present above the detection limits at 65% of sites. Concentrations of aluminum, iron, sodium, potassium, magnesium, and calcium, were particularly high at all sites. Aluminum, sodium, and iron concentrations greatly exceeded the EPA secondary drinking water standards at all sites. ^ Conclusion. The recreational water along Cypress Creek is impacted by wastewater from both permitted and non-permitted outfalls, which deposit inorganic substances into the water. Although a number of inorganic contaminants were present in the water, toxic metals regulated by the EPA were mostly below the recommended limits. However, high concentrations of aluminum, sodium, and iron in the Cypress Creek bring forward the issue of unauthorized discharges of salt water from mining, as well as industrial and domestic wastewater.^