Carboxylmethylation affects the proteolysis of myelin basic protein by Staphylococcus aureus V8 proteinase.

Bovine myelin basic protein (MBP), charge isoform 1 (C1) was carboxylmethylated by the enzyme D-aspartyl/L-isoaspartyl protein methyltransferase (EC. 2.1.1.77) and the carboxylmethylated protein was subjected to proteolysis by sequencing grade staphylococcal V8 proteinase at pH 4.0 to identify its carboxylmethylated modified aspartate and/or asparagine residues which are recognized by this methyltransferase. Native MBP, C1 was treated similarly and the proteolysis products were compared, using electrophoretic, chromatographic and amino acid sequencing techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in the kinetics of proteolysis between the native and the carboxylmethylated MBP, C1 which were confirmed using HPLC. Partial sequencing of the native and carboxylmethylated fragments eluting at about 29 min (P29) revealed cleavage of native MBP, C1 at Gly-127-Gly-128 and of the carboxylmethylated MBP, C1 at Phe-124-Gly-125. Additional evidence including tryptic subdigestion of carboxylmethylated P29 disclosed the following partial sequence for this peptide: Gly-Tyr-Gly-Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser-Ala-His-Lys-Gly-Leu-Lys- Gly-His-Asp-Ala-Gln-Gly-Thr-Leu-Ser-Lys-Ileu-Phe-Lys-. This sequence matches MBP residues 125-154. As a result of these findings, Asp-132 and Asp-144 were identified as two of the modified (isomerized or racemized) methyl-accepting L-aspartates in MBP. The results of the proteolysis experiments wherein the sequencing grade staphylococcal V8 proteinase was used at the rarely tested pH of 4.0, rather than at its commonly tested pH of 7.8, also disclose that the proteinase totally failed to recognize and hence cleave the two Glu-X bonds (Glu-82-Asn-83 and Glu-118-Gly-119) of MBP, preferring to cleave the protein at a number of hitherto unreported sites.

The carboxylmethylation of cerebral membrane-bound proteins increases with age.

Recently, we have characterized a membrane-bound (mb) component of brain protein carboxylmethyltransferase II (PCMT) which effectively carboxylmethylates endogenous mb methyl-accepting proteins (MAPs). (Neurochem. Int., 10 (1987) 155). We have also shown that exposing mb-MAPs to mild alkali leads to a marked increase in their recognition by PCMT. Since one of the likely consequences of the alkaline treatment appears to be the deamidation of selected protein-bound asparagines or aspartates, followed by the formation, in their place, of D-or L-isoaspartates, it is reasonable to assume that mb-MAPs constitute unique targets for the mb-PCMT because they contain such unnatural aspartate residues. Testing the relevance of this notion to the aging of cerebral mb-MAPs we focus in this report on age-related changes involving mb-MAPs. When two-or six-times washed (in 50 mM NaPO4 buffer, pH 6.5) 17,500 g, 30-min membranes or Percoll-gradient purified synaptic membranes were prepared from young (3-4 months) and old (11-12 months) rat brains and were incubated with 20 microM [3H]methyl S-adenosyl-L-methionine at pH 6.0, mb-MAP carboxyl[3H]methylation was significantly more intense in the old than in the young membranes, no additional increase being noted at 28-35 months. Mb-MAP carboxylmethylation increases were confirmed over a wide range of membrane protein concentrations and incubation times and are taken to reflect age-related modifications of the primary structure of susceptible mb-MAPs. To investigate these, we incubated young and old membranes, as well as their Lubrol-Px (1%) extracts (30 min, 0 degree C), with 0.05 M NH4OH for 90 min at 37 degrees C, a treatment which left PCMT activity largely unaffected. Our findings reveal that the effect of the NH4OH treatment on the generation of carboxylmethylatable sites was markedly smaller in "old" than in "young" proteins, suggesting that "new" carboxylmethylatable sites are generated in susceptible mb-MAPs in situ, by a process accompanying, or otherwise marking, the natural aging of neural membrane proteins.

The characterization of a membrane-bound protein carboxylmethylation system in brain.

The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein carboxylmethylation system is shown to include, in addition to nerve cell bodies and purified synaptosomes, astrocytes and oligodendroglia. The membrane-bound nature of the protein carboxylmethylation system was investigated and these studies revealed a tight association which exposure to several detergents could only partially solubilize. The membrane-bound PCMT could be shown to undergo activation after treatment with Na-deoxycholate and CHAPs, while after its detergent-induced solubilization PCMT activation was observed after Na-deoxycholate, Nonidet P-40 and Lubrol-P(X). Solubilization of the carboxylmethylation system in CHAPS appeared to be more effective at 0 degrees C than at 25 degrees C or 37 degrees C. Detergent treatment was shown to be deleterious to the MAPs as PCMT substrates, particularly when the exposure was extended to more than 1 h. These observations prompted exposure of the brain membranes and of their Lubrol-P(X) and Nonidet P-40 extracts to NH(4)OH, treatment which promotes the conversion of protein asparagine residues to atypical l-isoaspartate residues, recently shown (in synthetic peptides) to be the single most effective residue recognized for carboxylmethylation by PCMT. We found up to a 400% enhancement of the carboxylmethylation of solubilized membrane MAPs by the equally solubilized PCMT (which resisted the alkaline treatment virtually unscathed) after 90 min at 37 degrees C in 0.05 M NH(4)OH. However, when brain membrane Lubrol-P(x) extracts were first subjected to bis(I,I-trifluoroacetoxy)-iodobenzene, a reagent which converts the carboxyamide group of protein-bound asparagine to the corresponding primary amine, the amount of MAPs susceptible to be acted upon by 0.05 M NH(4)OH became greatly reduced. Finally, acidic slab gel electrophoresis of membrane-bound MAPs, carboxyl-[(3)H]-methylated by the membrane-bound PCMT, revealed the presence of about 12 radioactive protein bands, ranging in MW from under 20 KDa to about 90 KDa.

Cerebellar tRNA methyltransferases: a developmental study.

Developmental patterns of homologous and heterologous tRNA methylation by cerebellar tRNA methyltransferases are described. The study revealed that: (a) homologous tRNA methylation results in the predominant formation of N2-methylguanine and 1-methyladenine; (b) tRNA methyltransferase of bulk-isolated Purkinje and granule cells methylate E. coli tRNAglu2 in vitro in a characteristic manner, and (c) the methylation of 8-day-old cerebellar, cortical and hepatic tRNA in vivo yields tRNAs containing different proportions of methylated bases. The findings suggest that the presumably cell-specific populations of cerebellar tRNA methyltransferases continue to alter their substrate recognition characteristics up to and beyond the first month of post-natal life.

Beta-alanine uptake is not a marker for brain astroglia in culture.

The properties of the beta amino acid transport system were examined in cultivated rat brain astrocytes, using the specific probe, beta-alanine. The uptake of beta-alanine is thought to be glial specific. Beta-alanine was not actively transported and the intracellular accumulation was not altered by coincubation with GABA, alanine, glutamate, or methionine. We suggested therefore that beta-alanine is passively taken up by a mechanism distinct from the transport system for GABA.

Cerebral methylations in epileptogenesis.

This chapter deals with the neurochemical consequences of the administration of the chemical convulsant agent L-methionine-dl-sulfoximine (MSO) on the brain of rodents. The principal notion is that this convulsant agent differs qualitatively from most quick-acting and predominantly lethal convulsant agents, commonly used in laboratory studies in epilepsy modeling because it has a preconvulsant latency period of several hours and also because it need not be fatal to the animals receiving it if they are properly managed during the preconvulsant period and following the first seizure attack. The historical profile of MSO as a useful and unique laboratory tool for neurochemical and molecular studies is briefly recounted, and the point is made that MSO is a close derivative of the amino acid L-methionine, which is used by each and every brain cell as a protein building block and as a precursor of the universal cellular methyl donor molecule, S-adenosyl-L-methionine. The importance of methylations, a set of reactions which consist in the transfer of the methyl group of S-adenosyl-L-methionine to several dozens of endogenous methyl acceptor molecules, small and large, is stressed and reviewed both historically and as this process relates to MSO epileptogenesis. The multiple effects of MSO on the methylation of small MW compounds, histamine being the working example, are reviewed. The involvement of phospholipid, nucleic acid, and protein methylations, all apparent targets of MSO action, in that they respond to the MSO challenge by a significant rate increase is elucidated. Finally, the effects of MSO at the functional level of brain receptor action are presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in activity in cerebral methyltransferases and monoamine oxidases between audiogenic seizure susceptible and resistant mice and deermice.

The specific acitivity of cerebral histamine N-methyltransferase (HMT) was significantly lower in the audiogenic seizure-susceptible (SS) 21-day old DBA/2J mouse when compared to the non-susceptible 70-day old DBA/2J mouse but not when compared to the seizure resistant (SR) C57B1/6J mouse at 21 days of age. There was no significant difference between the two strains at 70 days of age. The lower HMT specific activity was also observed in a SS mutant of the deermouse Peromyuscus maniculatus, relative to the SR, wild-type animal. The activity of cerebral catechol-O-methyltransferase (COMT) was significantly lower in the DBA/2J mice relative to the C57B1/6J at 21 and 70 days while, in Peromyscus, it was higher in the SS mutant than in the SR animal. The activity of MAO, B was lower in the 21-day old, relative to the 70-day old DBA/2J and the 21-day old C57B1/6J mice. There were no differences in MAO A or B between SS and SR Peromyscus. The findings raise the possibility that cerebral methylation may operate at characteristic rates in SS animals.