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Hydrogen sulfide, H2S, is the third endogenous gas with cardiovascular properties, after nitric oxide and carbon monoxide. H2S is a potent vasorelaxant, and its deficiency is implicated in the pathogenesis of hypertension and atherosclerosis. Cystathionine beta-synthase, cystathionine gamma- lyase, and 3-mercaptopyruvate sulfurtransferase catalyze H2S formation. Chronic kidney disease is characterized by high prevalence of hyperhomocysteinemia, hypertension, and high cardiovascular mortality, especially in hemodialysis patients. H2S levels are decreased in hemodialysis patients through transcriptional deregulation of genes encoding for the H2S-producing enzymes. Potential implications relate to the pathogenesis of the manifestations of the uremic syndrome, such as hypertension and atherosclerosis. (C) 2010 by the National Kidney Foundation, Inc. All rights reserved.

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: Implications for mechanism.BackgroundSeveral alterations of protein structure and function have been reported in uremia. Impairment of a transmethylation-dependent protein repair mechanism possibly related to a derangement in homocysteine metabolism is also present in this condition, causing erythrocyte membrane protein damage. Homocysteine may affect proteins via the accumulation of its parent compound S-adenosylhomocysteine (AdoHcy), a powerful in vivo methyltransferase inhibitor. However, since plasma homocysteine is mostly protein bound, a direct influence on protein structures cannot be ruled out. We measured the levels of L-isoaspartyl residues in plasma proteins of uremic patients on hemodialysis. These damaged residues are markers of molecular age, which accumulate when transmethylation-dependent protein repair is inhibited and/or protein instability is increased.MethodsL-isoaspartyl residues in plasma proteins were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). Plasma concentrations of homocysteine metabolites were also measured under different experimental conditions in hemodialysis patients.ResultsThe concentration of damaged plasma proteins was increased almost twofold compared to control (controls 147.83 ± 17.75, uremics 282.80 ± 26.40 pmol of incorporated methyl groups/mg protein, P < 0.003). The major protein involved comigrated with serum albumin. Although hyperhomocysteinemia caused a redistribution of thiols bound to plasma proteins, this mechanism did not significantly contribute to the increase in isoaspartyl residues. The S-adenosylmethionine (AdoMet)/AdoHcy concentration ratio, an indicator of the flux of methyl group transfer, was altered. This ratio was partially corrected by folate treatment (0.385 ± 0.046 vs. 0.682 ± 0.115, P < 0.01), but protein L-isoaspartate content was not.ConclusionsPlasma protein damage, as determined by protein L-isoaspartyl content, is increased in uremia. This alteration is to be ascribed to an increased protein structural instability, rather than the effect of hyperhomocysteinemia.

We have purified the more acidic major isozyme (II) of the human erythrocyte L-isoaspartyl/D-aspartyl methyltransferase and compared its structure to that of the previously sequenced isozyme I. These isozymes are both monomers of 25,000 molecular weight polypeptides and have similar enzymatic properties, but have isoelectric points that differ by one pH unit. Analysis of 16 tryptic peptides of isozyme II accounting for 89% of the sequence of isozyme I revealed no differences between these enzyme forms. However, analysis of a Staphylococcal V8 protease C-terminal fragment revealed that the last two residues of these proteins differed. The Trp-Lys-COOH terminus of isozyme I is replaced by a Asp-Asp-COOH terminus in isozyme II. Southern blot analysis of genomic DNA suggests that the human genome [corrected] may contain only a single gene encoding the enzyme. We propose that the distinct C-termini of isozymes I and II can arise from the generation of multiple mRNA's by alternative splicing.

Endoproteinase Asp-N, a metalloprotease from a mutant strain of Pseudomonas fragi, has been reported to specifically cleave on the N-terminal side of aspartyl and cysteic acid residues. We utilized this enzyme to generate fragments for determining the amino acid sequence of the D-aspartyl/L-isoaspartyl methyltransferase isozyme I from human erythrocytes. Surprisingly, we identified cleavage sites for this enzyme at the N-terminal side of several glutamyl residues in addition to the expected cleavage sites at aspartyl residues. The ability of this enzyme to cleave polypeptides at both glutamyl and aspartyl residues was confirmed by mapping additional sites on erythrocyte carbonic anhydrase I. These results indicate that a more appropriate name for this enzyme may be Endoproteinase Asp/Glu-N.