Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.)

Summary Differences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.Paper No. 9498 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Enkephalin pseudopeptides: resistance to in vitro proteolytic degradation afforded by amide bond replacements extends to remote sites

Five analogs of leucine enkephalin containing the CH2S group as an amide bond replacement were evaluated with respect to resistance toward degradation by human serum in an HPLC-based assay using both ultraviolet and electrochemical detection. Analogs with the modification at the 1-2, 2-3, 3-4, or 4-5 peptide linkages demonstrated half-lives of 118, 85, 134, and 318 min vs. 12 min for the parent peptide. A pseudopeptide analog with additional D-Ala2 protection had a half-life of greater than 1000 min, while the potent [D-Ala2]-leucine enkephalin analog showed approximately a 10-fold increase in stability. The significant increase in stability for a compound with protection only at the C-terminus suggests that serum enzymes may have greater specificity toward backbone changes than previously realized.     

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).