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A monoclonal antibody designated III 5H8 was shown to bind both to astrocytes and to myelin sheaths as studied with immunocytochemical techniques on brain sections and cell cultures. Binding to astrocytes was confirmed by double immunofluorescent labelling of frozen sections and cell cultures with anti-GFAP, and appeared to be sensitive to formalin treatment. Binding to myelin sheaths was confirmed by comparing sections labelled with III 5H8 with sections labelled with antibodies against axons and myelin basic protein as well as by staining of sections of hypomyelinated spinal cord with III 5H8. On immunoblots of separated white matter III 5H8 revealed two bands, while on immunoblots of purified myelin only one band was seen. The findings are discussed with respect to the function of astrocytes in white matter and shared antigenic determinants between astrocytes and oligodendrocytes.

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that possess phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that phosphorylated neurofilaments are protected against proteolysis, and dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.