Our exhaustive review of the many papers on brain proteinases led us to choose calcium activated neutral proteinases (CANP I, II and III) as the brain enzymes that we will investigate. CANP I is located in the neurons, CANP II is located in the glia cells, and CANP III has not been associated with a particular structure. Presently, we are developing the specific analytical methods needed to extract, purify, characterize and assay the CANP's. Our previous investigation of human myelin basic protein (HBP) led to the identification and characterization of a new form of HBP with a molecular weight of 17.2 kDa and highly purified HBP-component 1, the unmodified 18.5 kDa protein. Highly purified HBP-component 1 was cleaved with thrombin and the resulting two peptides (1-97 & 98-170) were purified and characterized. HBP-component 1, 17.2 kDa HBP and the two thrombic peptides (residues 1-97 & 98-170) were used in collaboration with Dr. John Richert, Dept. of Neurology, Georgetown University. Forty myelin basic protein-reactive T cell clones were isolated from a multiple sclerosis patient and used to identify human T cell recognition sites on the HBP molecule. At least three sites have been identified: One in the N-terminal half of the molecule (residues 1-97), one in the C- terminal (residues 98-170), and one which spans residues 97-98. In collaboration with Dr. Audrey Stone, we have completed the investigation on the role of phosphorylation on the conformational adaptability of bovine myelin basic protein (MBP). The limited digestion of MBP-components 2 + 3 yielded a mono-phosphorylated component which was phosphorylated only at threonine 97. From the circular dichroism of this homogenous mono-phosphorylated MBP, we determined that the beta- structure of this protein was increased by 7%, when compared with MBP-component 1. The single phosphorylation on threonine 97 produced a change in the macrostructure of the protein involving about 12 amino acid residues.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Intramural Research (Z01)
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U.S. National Institute of Mental Health
United States
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