An RNA homologous to U2 RNA and a single copy gene encoding the RNA homolog have been characterized in the microsporidian, Vairimorpha necatrix. The RNA which is 165 nucleotides in length possesses significant similarity to U2 RNA, particularly in the 5' half of the molecule. The U2 homolog contains the highly conserved GUAGUA branch point binding sequence seen in all U2 RNAs except those of the trypanosomes. A U2 RNA sequence element implicated in a U2:U6 RNA intermolecular pairing is also present in the U2 homolog. The V.necatrix U2 RNA homolog differs at positions previously found to be invariant in U2 RNAs and appears to lack an Sm binding site sequence, The RNA can be folded into a secondary structure possessing three of the four principal stem-loops proposed for the consensus U2 RNA structure. A cis-diol containing cap structure is present at the 5' end of the U2 homolog. Unlike the cap structures seen in U-snRNAs and mRNAs it is neither 2,2,7-trimethylguanosine, gamma-monomethyl phosphate, nor 7-methylguanosine.

To gain insight into possible cellular protective mechanisms against the insult of formaldehyde, we have investigated this molecule's reactivity with both naturally occurring thiol compounds including glutathione and L-ascorbic acid. By UV measurements, formaldehyde was found to rapidly react with glutathione forming an S-hydroxymethyl covalent adduct. The adduct which was confirmed by NMR is transiently stable. Formaldehyde is also significantly reactive with L-ascorbic acid in a reaction which was observed to be dissimilar to its reaction with dimedone. The reaction of formaldehyde with glutathione was reduced by 40% in the presence of an excess amount of L-ascorbic acid, due to the trapping of formaldehyde by L-ascorbic acid. The data suggest that L-ascorbic acid may have a possiblein vivo role in the metabolism of formaldehyde, thereby protecting cellular glutathione from possible depletion.

This chapter describes protein lysyl methylation enzymology of cytochrome c methylases with emphasis on the enzyme characterized from Neurospora crassa. The enzymatic transfer of radioactive methyl groups from S-adenosyl-L-[methyl-14C]methionine into trichloroacetic acid (TCA)-precipitable material (including cytochrome c) is determined under conditions which have been generally found to be favorable for the methylation of ɛ-amino groups of lysyl residues (pH 9.0). Enzyme activity is expressed as picomoles of [14C] methyl incorporated per minute per milligram of enzyme protein (specific activity). When the 3500-fold purified enzyme is run on sodium dodecyl sulfate-polyacrylamide gels which have been subsequently stained with Coomassie blue and scanned at 560 nm, only two bands are apparent on the scanning profile with one comprising four times the staining intensity of the other. Thus, the preparation is judged to be 80% homogenous. The enzyme is stable for several months when stored in the presence of 50% saturated ammonum sulfate at 0-4°.

The patterns of membrane protein carboxyl-methylation by protein methylase II (S-adenosylmethionine: protein-carboxyl O-methyltransferase, EC 2.1.1.24) in intact human erythrocytes were shown to differ markedly whether the methyl donor, S-adenosyl-L-[methyl-3H]methionine, was supplied exogenously or formed intracellularly via exogenously added L-[methyl-3H]methionine. The differences include the following. (1) The methylation of cytoskeletal components (band 2.1 and 4.1) occurs only in the case of the L-[methyl-3H]methionine-labelled cells. (2) The methionine-mediated methylation was much less sensitive to S-adenosyl-L-homocysteine inhibition than the adenosylmethionine-mediated methylation (22% versus 95% inhibition at 10 microM). (3) The membrane protein methylation mediated by exogenous adenosylmethionine and methionine differed markedly in their alkali labilities; at pH 6.0, 30% of the adenosylmethionine-mediated protein methyl esters were hydrolysed after 30 min (37 degrees C) while the methionine-mediated esters were stable. At pH 7.4, the respective labilities were 60% and 30% for the 30 min incubation. To explain these results, a possible involvement of cytoskeletal structure associated with the intact erythrocyte is discussed.

Methylation of horse heart cytochrome c has been examined in vitro with [methyl-14C]methanesulfonate (MMS) and [1-methyl-14C]-1-nitrosourea (MNU) as alkylating agents. Analysis of protein hydrolyzates by an automatic amino acid analyzer indicates that, at pH 9.0 with MMS, ϵ-N-monomethyllysine is found to be the only major methylated basic amino acid. On the other hand, the identity of the predominant basic amino acid residue which is [methyl-14C]-labeled by MNU cannot be determined at present. Peptide mapping of chymotryptic digests of cytochrome c after reaction with MMS reveals a lack of specificity in methylation of a specific lysine residue in this hemoprotein.

The cytochromes c of plants (e.g., wheat germ) possess two trimethyllysines, residues 72 and 86. In order to investigate the nature of these methylations, we have purified a cytochrome c specific methylase S-adenosylmethionine: protein(lysine) N-methyltransferase (protein methylase III) from wheat germ 135-fold. The in vitro site of methylation by both the purified enzyme and crude wheat germ extract toward various forms of horse heart cytochrome c was localized by two dimensional peptide mapping, Aminex A-5 column peptide analysis, and CNBr cleavage analysis to be the residue 72 lysine. However, no additional sites, in particular residue 86, were seen to be methylated. Although the enzyme is highly specific toward cytochrome c as an in vitro protein substrate, avian cytochromes c are seen to be much better substrates than those from mammalian sources. The enzyme possesses an extremely low Km for apocytochrome c (1.21 microM), suggesting that methylation may occur before heme attachment in vivo. Various S-adenosyl-L-homocysteine analogues were tested for their inhibitor capability toward the enzyme; it was observed that only the D and L forms of S-adenosylhomocysteine are inhibitors while analogues modified in the adenine or homocysteine moieties do not possess inhibitory capability. Results from the Aminex A-5 column chromatography of horse heart cytochrome c chymotryptic digest showed the N epsilon-methyl-, N epsilon-dimethyl-m and N epsilon-trimethyllysine forms of the residue 68-74 peptide to elute earlier than the unmethylated form. This results suggest that the methylated peptides are less basic than the unmethylated form.

The in vivo stability of methylated and unmethylated cytochrome c in Saccharomyces cerevisiae was studied by pulse-labeling the hemoproteins with [methyl-3H]-methionine and/or [2-14C]methionine and following the fate of these proteins under anaerobiosis and in the presence of cycloleucine. These two conditions will respectively block further cytochrome c synthesis and inhibit methylation by lowering the cellular S-adenosyl-L-methionine pool and, thus, permit an unambiguous interpretation of the data. The results showed that the rate of degradation of unmethylated cytochrome c was constant throughout the chase period, while methylated cytochrome c degradation was seen only in the later part of cold chase. At the end of the chase period (40 h), the extent of degradation of the unmethylated species was three times higher than the methylated species. This indicated that the methylation of cytochrome c has a protective effects against the intracellular proteolytic enzyme attack on itself. Furthermore, this protective effect was considerably reduced in the petite mutant, which lacks high affinity cytochrome c binding sites, functional cytochrome c reductase, and oxidase, and possesses a less integrated and organized mitochondrial membrane. These results led us to the conclusion that the mechanism of methylated cytochrome c stabilization is best explained by a higher efficacy of binding to the mitochondria.

A highly purified protein methylase III from Neurospora crassa or Saccharomyces cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c. The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing. Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively. Since methyl substitution increases the basicity associated with the ϵ-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase. Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the ϵ-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome c. The enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule. This paper alsodeals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique.

chromes c from fungal, plant, and protozoan eukaryotic organisms have been found to possess 1 or more residues of this amino acid (2).In contrast, none of the cytochromes c of the animal kingdom (either vertebrate or invertebrate) possess E-N-methylated lysines (2).Fungal cytochromes c from such organisms

A simple method to prepare small quantities of radioactive ε-N-methylated lysines for use as analytical standard markers, which does not involve elaborate organic synthesis, is highly desired in numerous research applications. The method in this report involved growingNeurospora crassa orSalmonella typhimurium in the presence of uniformly labeled [U-14C] lysine or reductively methylating a protein with [14C]formaldehyde at pH 9.0. The radiolabeled proteins were then isolated and hydrolyzed in 6n HCl. Finally, the radioactive methylated lysines in the acid hydrolysates were isolated by Dowex 50 column chromatography.