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Currently, there is no biochemical test to assist in the behavioral diagnosis of autism. We observed that levels of phosphatidylethanolamine (PE) were decreased while phosphatidylserine (PS) were increased in the erythrocyte membranes of children with autism as compared to their non-autistic developmentally normal siblings. A new method using Trinitrobenezene sulfonic acid (TNBS) for the quantification of PE and PS (amino-glycerophospholipids, i.e., AGP) in the plasma of children was developed and standardized. Wavelength scans of TNBS-PE and TNBS-PS complexes gave two peaks at 320 nm and 410 nm. When varying concentrations of PS and PE were used, a linear regression line was observed at 410 nm with TNBS. Using this assay, the levels of AGP were found to be significantly increased in the plasma of children with autism as compared to their non-autistic normal siblings. It is proposed that plasma AGP levels may function as a potential diagnostic marker for autism.

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: 1) each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; 2) when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68kDa protein by CANP2 and CANP3 was noted whereas 210kDa and 150kDa proteins remained largely intact. CANP1 degraded the 150kDa and 68kDa proteins more rapidly than 210kDa protein, where only a slight effect was noted. These data provide further proof of the existance of three different forms of CANP in the brain, and indications of the resistance of 210kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.

A calcium-activated neutral protease (CANP) has been purified 2,800 fold, to near homogeneity, from human platelets. The purification procedure involved ammonium sulfate fractionation of the platelet cytosol followed by chromatography on Sephacryl S-200, DEAE-Sephacel, Agarose-Hexylamine, Agarose-Octylamine and alpha-casein-Sepharose 4B affinity gel. The protease consisted of two polypeptides of Mr = 74,000 and 28,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzed [methyl-14C] alpha-casein at a significant rate of 37 degrees C which was, therefore, used as an exogenous substrate. Microtubules and intermediate filament proteins were also susceptible to hydrolysis by the purified protease. It attained maximum activity at 0.06 uM CaCl2 and displayed two pH maxima: one at 5.5 and the other at 6.5. The protease was fully active in the presence of MnCl2 and was about 75% active with BaCl2 and SrCl2. Among the actinomycete protease inhibitors, leupeptin, antipain and pepstatin, the order of inhibition was: leupeptin greater than antipain greater than pepstatin. The protease was also inhibited by sulfhydryl modifying agents.

A Ca 2+ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.