Veniamin N. Lapko

In vivo carbamylation and acetylation of water-soluble human lens αB-crystallin lysine 92

Several post‐translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an α‐crystallin isolated from the water‐soluble portion of a cataractous human eye lens indicated that it was a modified αB‐crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the sample was structurally heterogeneous. Lys 92 was acetylated in one population and carbamylated in another. Although carbamylation of lens crystallins has been predicted, this is the first documentation of in vivo carbamylation of a specific site. These results are also the first documentation of in vivo lysine acetylation of αB‐crystallin. Both modifications alter the net charge on αB‐crystallin, a feature that may have significance to cataractogenesis.

Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide

Derivatization of cysteinyl residues is often used to prevent the formation of disulfide bonds during protein isolation and analysis. The most commonly used reagents are iodoacetic acid and iodoacetamide, which increase the molecular mass of the protein by 58 or 57 Da, respectively, for each derivatized cysteine. A possible side reaction is derivatization of methionine. In our analysis of derivatized human lens alphaA-crystallins, we found an apparent molecular mass 48 Da lower than the mass expected for alphaA-crystallin with the cysteines carboxyamidomethylated. Analysis of a tryptic digest of this protein showed that both cysteines and one methionine had been derivatized. Peaks indicating a molecular mass 48 Da less than expected for the protein with only cysteines derivatized were attributed to fragmentation of the derivatized methionine through collision-induced dissociation in the electrospray ionization source. An awareness of this artifact is important to investigators searching for proteins and their modified forms in complex mixtures.

Methylation and carbamylation of human γ‐crystallins

Accessible sulfhydryls of cysteine residues are likely sites of reaction in long‐lived proteins such as human lens crystallins. Disulfide bonding between cysteines is a major contributor to intermolecular cross‐linking and aggregation of crystallins. A recently reported modification of γS‐crystallins, S‐methylation of cysteine residues, can prevent disulfide formation. The aim of this study was to determine whether cysteines in γC‐, γD‐, and γB‐crystallins are also S‐methylated. Our data show that all the γ‐crystallins are S‐methylated, but only at specific cysteines. In γD‐crystallin, methylation is exclusively at Cys 110, whereas in γC‐ and γB‐crystallins, the principal methylation site is Cys 22 with minor methylation at Cys 79. γD‐crystallin is the most heavily methylated γ‐crystallin. γD‐Crystallins from adult lenses are 37%–70% methylated, whereas γC and γB are ∼12% methylated. The specificity of γ‐crystallin methylation and its occurrence in young clear lenses supports the idea that inhibition of disulfide bonding by S‐methylation may play a protective role against cataract. Another modification, not reported previously, is carbamylation of the N termini of γB‐, γC‐, γD‐crystallins. N‐terminal carbamylation is likely a developmentally related modification that does not negatively impact crystallin function.

Modifications of human βA1/βA3‐crystallins include S‐methylation, glutathiolation, and truncation

Disulfide bonding of lens crystallins contributes to the aggregation and insolubilization of these proteins that leads to cataract. A high concentration of reduced glutathione is believed to be key in preventing oxidation of crystallin sulfhydryls to form disulfide bonds. This protective role is decreased in aged lenses because of lower glutathione levels, especially in the nucleus. We recently found that human γ‐crystallins undergo S‐methylation at exposed cysteine residues, a reaction that may prevent disulfide bonding. We report here that βA1/A3‐crystallins are also methylated at specific cysteine residues and are the most heavily methylated of the human lens crystallins. Among the methylated sites, Cys 64, Cys 99, and Cys 167 of βA1‐crystallin, methylation at Cys 99 is highest. Cys 64 and Cys 99 are also glutathiolated, even in a newborn lens. These post‐translational modifications of the exposed cysteines may be important for maintaining the crystallin structure required for lens transparency. Previously unreported N‐terminal truncations were also found.

A SIMPLE AND IMPROVED METHOD OF ISOLATION AND PURIFICATION FOR NATIVE OAT PHYTOCHROME

Abstract— A simple procedure for the isolation and purification of 124 kDa phytochrome (phyA) from etiolated Avena seedlings has been developed employing ammonium sulfate back‐extraction. After solubilization of the ammonium sulfate precipitate (250 g/L) an additional ammonium sulfate fractionation with 17 g per 100 mL rather than column chromatography was performed. After several steps of the “washing‐out” procedure with 100 mM phosphate buffer, phytochrome was solubilized in 10 mM phosphate buffer. The resulting phytochrome had a specific absorbance ratio (SAR = A666/A28o) ranging from 0.60 to 0.85. These values are equivalent to those of phytochrome preparations after hydroxyla‐patite chromatography‐ammonium sulfate back‐extraction. The total isolation‐purification time was 8 h and yield of the chromoprotein was 50% higher than the yield using conventional techniques. The phytochrome preparation, after application to a Toyopearl HW‐65S gel filtration column, produced very pure 124 kDa phyA with a specific absorbance ratio greater than 1.00. The spectral characteristics are identical to those described for the best of the highly purified native chromoprotein preparations.

Expression of βA2-crystallin in human lenses

Evidence of betaA2-crystallin expression has been detected in human lenses. The protein, which co-elutes with betaA1/A3 from reversed phase HPLC separation of beta(L)-crystallins, accounts for 1-2% of the lens crystallins. Its molecular mass, M(r) 22 006, is consistent with the cDNA deduced sequence with addition of acetylation at the N-terminal serine residue. Approximately 20% of the protein is phosphorylated at Ser30.

Primary Structure of Ferredoxin from Bovine Kidney Mitochondria

Kidney mitochondrial ferredoxin (renodoxin) is a component of the cytochrome P-450-dependent enzymatic system whose main function is the hydroxylation of vitamin D3 in the 1a- and 24-positions. The complete amino acid sequence of renodoxin was determined by protein chemistry and mass spectrometry. The mature renodoxin has 128 amino acid residues. The N- and C-terminal regions of renodoxin are subject to proteolytic modification, this being the origin of heterogeneous molecular mass (from 14,200 to 12,400 kD) of purified protein preparations. The antigenic structure of renodoxin was studied using antibodies to peptide fragments of a homologous protein, adrenodoxin.