Cinda-Sue Davis

Neurotoxic esterase: Characterization of the solubilized enzyme and the conditions for its solubilization from chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents

Neurotoxic esterase (NTE) is a membrane-bound protein found in highest concentration in brain and lymphocytes. The enzyme has no known physiological function, but its organophosphorylation and aging in neural tissue are thought to trigger the pathogenesis of organophosphorus-induced delayed neuropathy (OPIDN). Solubilization of NTE from microsomal membranes from hen or chick brain was studied with ten detergents encompassing ionic, zwitterionic, or nonionic types. Corrected yields of NTE solubilized over a range of [detergent]/[protein] ratios were determined by dividing the activity not sedimenting in detergent at 100,000 g for 60 min at 4 degrees by the activity in the original microsomal fraction with no detergent present. Highest corrected yields were obtained with sodium cholate (44%), Triton X-100 (48%), and nonyl-GPS (57%). Partial loss of NTE activity occurred in the presence of detergent which could be prevented by the inclusion of asolectin in the solubilization preparation. NTE could not be solubilized by omitting detergent or by substituting 2 M NaCl for detergent. Mipafox pI50 values obtained from complete titration curves carried out on NTE solubilized in Triton X-100, sodium cholate, or sodium cholate/asolectin were indistinguishable from the value for native enzyme from brain homogenate. These results indicate that NTE exhibits the properties of an integral membrane protein with lipid dependence. The enzyme can be solubilized in good yield with a variety of detergents with retention of its characteristic differential inhibition by paraoxon and mipafox, a necessary prelude to bulk purification of the enzymatically active protein.

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.

Kinetics of heat inactivation of phenyl valerate hydrolases from hen and rat brain.

Heat inactivation was studied at 45, 50, 55, and 60 degrees for all of the phenyl valerate hydrolases (PVase), including neurotoxic esterase (NTE) and inhibitor-resistant esterase (IRE), in homogenates of hen or rat brain or in preparations of hen brain microsomal membranes. Hen and rat brain homogenates were prepared in buffer (50 mM Tris/0.20 mM EDTA, pH 8.00, at 25 degrees). Hen brain microsomes were suspended either in buffer or in aqueous dimethyl sulfoxide (DMSO, 40%, w/v), or solubilized either in aqueous Triton X-100 (0.10%, w/v) or in 40% (w/v) DMSO. Enzyme activities were measured at 37 degrees using phenyl valerate as substrate. Each enzyme activity in all of the preparations exhibited biphasic heat inactivation kinetics. Apparent rate constants were calculated for the fast (kf) and slow (ks) reactions, along with the relative amounts of activity in each component (Af, As) expressed as percentages of the total activity. For a given preparation and temperature, respective values of kf or ks were similar for PVase, NTE, and IRE, with a mean kf/ks ratio of 52 across all preparations. Af and As were a function of temperature. Mean values of the apparent activation energies (Ea) for all activities and preparations were 44 and 25 kcal/mol for the fast and slow inactivation reactions respectively. These results indicate that all phenyl valerate hydrolases in hen and rat brain undergo a common heat-induced structural change leading to loss of enzymic activity.

A rapid sensitive assay for phosphatidate phosphohydrolase

For a purified preparation of the soluble form of phosphatidate phosphohydrolase (EC 3.1.3.4) from guinea pig cerebral cortex, I-O-alkyl-racglycerol 3-phosphate was found to be accepted as a substrate. This substrate analog was tritium-labeled in order to serve in a rapid sensitive assay for the enzyme, in which labeled I-alkyl glycerol is released. Heat denaturation and enzyme activity dependence on pH indicated that I-O-alkyl-rat-glycerol 3phosphate phosphohydrolase and phosphatidate phosphohydrolase activities in the preparation are attributable to the same enzyme. I-O-Alkyd-rat-glycerol 3phosphate was hydrolyzed with a

Solubilization of hen brain neurotoxic esterase in dimethylsulfoxide.

Neurotoxic esterase is the putative site of initiation of organophosphorus-induced neuropathy. The protein is membrane-associated and will thus require solubilization before it can be purified. Its enzymic activity was retained in hen brain microsomes suspended in 10-60% (v/v in water) dimethylsulfoxide and 5-20% dimethylacetamide, but lost in 5-20% 1- and 2-propanol as well as higher concentrations of dimethylsulfoxide. Soluble activity (100,000 x g, 60 min supernatant) was not obtained with dimethylacetamide, but 24% of the recovered activity (67%) was solubilized in 40% dimethylsulfoxide, with retention of its native response to inhibitors. Solvent extraction of active enzyme is of intrinsic interest and expands the options for its purification.

The University of Michigan's M-STEM academies program: Examining the social community of future engineers

A social community framework is used to examine the program elements of the University of Michigans (UM) M-Engin Program, an undergraduate engineering mentoring program, to provide insights about its social community elements. At this initial research stage, a methodical analysis of the program data (e.g., analytics, features, elements, and artifacts) was performed to create a portfolio to begin to understand the role of social community within the M-Engin Program. From this research, we can learn how to better serve members of various program communities by identifying opportunities for improvement. Future research will include conducting informal interviews with the programs coordinators and surveying and interviewing program participants.