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When the concentrations of alpha-S100 (alpha subunit of S100 protein) and beta-S100 (beta subunit) proteins in various tissues of human and rat were determined by the immunoassay method, immunoreactive beta-S100 was present at high levels in the CNS, adipose tissue, and cartilaginous tissue. In contrast, the alpha-S100 was found in the heart and skeletal muscles at concentrations much higher than in the CNS. The concentration of alpha-S100 protein was also high in the heart and skeletal muscles of bovine, porcine, canine, and mouse. Since beta-S100 protein levels in those tissues were low, it was suggested that S100 protein in the muscle tissues is present mainly as the alpha alpha form (S100a0 protein). To confirm the above findings, immunoreactive alpha-S100 protein was purified from human pectoral muscle by employing column chromatographies with butyl-Sepharose, diethylaminoethyl (DEAE)-Sepharose, Sephadex G-75, and finally with an anion-exchange Mono Q column in a HPLC system. The elution profile of alpha-S100 protein from the Mono Q column suggested some heterogeneity of the final preparation. However, each of these fractions traveled with a single band at a position similar to that of bovine S100a0 protein on slab-gel electrophoresis. The amino acid composition of the final preparation was very similar to the composition of bovine S100a0 protein. The purified alpha-S100 protein was eluted from a gel-filtration column (Superose 12) in the same fraction as bovine S100a0 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) were prepared by use of beta-D-galactosidase from Escherichia coli as label and the purified rabbit antibodies to alpha alpha and gamma gamma enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen-coupled Sepharose. Sandwich-type immunoassay systems with the galactosidase-labeled antibody Fab' fragments and the antibody F(ab')2-immobilized polystyrene beads could determine amounts as small as 1 amol (10(-18) mol) of each isozyme. Purkinje cell bodies picked up from the bulk-separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the gamma gamma but also the alpha gamma and alpha alpha enolases at a level of amol per cell body, although the concentration of gamma gamma was the highest. Immunohistochemical experiments on te cerebellum with the peroxidase-labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both alpha and gamma subunits of hte enolase were stained intensely in axons.

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