Michail A. Alterman

Proteomic identification of age-dependent protein nitration in rat skeletal muscle.

Age-related protein nitration was studied in skeletal muscle of Fisher 344 and Fisher 344/Brown Norway (BN) F1 rats by a proteomic approach. Proteins from young (4 months) and old (24 months) Fisher 344 rats and young (6 months) and old (34 months) Fisher 344/BN F1 animals were separated by 2-D gel electrophoresis. Western blot showed an age-related increase in the nitration of a few specific proteins, which were identified by MALDI-TOF MS and ESI-MS/MS. We identified age-dependent apparent nitration of beta-enolase, alpha-fructose aldolase, and creatine kinase, which perform important functions in muscle energy metabolism, suggesting that the nitration of such key proteins can be, in part, responsible for the decline of muscle motor function of the muscle. Furthermore, we have identified the apparent nitration of succinate dehydrogenase, rab GDP dissociation inhibitor beta (GdI-2), triosephosphate isomerase, troponin I, alpha-crystallin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Fatty acid discrimination and ω-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein☆

The omega-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regionspecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strength enzyme-substrate interactions. To investigate the chemical basis for these features of omega-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of omega-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein omega-hydroxylated methyl laurate and lauryl alcohol about 1/10th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via omega-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the omega-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid omega-hydroxylation by 43% at equimolar concentrations. omega-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.

Quantitative Analysis of Cytochrome P450 Isozymes by Means of Unique Isozyme-Specific Tryptic Peptides: A Proteomic Approach.

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.

Amino Acid Analysis by Means of MALDI TOF Mass Spectrometry or MALDI TOF/TOF Tandem Mass Spectrometry

Here, we describe two different amino acid analysis protocols based on the application of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS). First protocol describes a MALDI TOF MS-based method for a routine simultaneous qualitative and quantitative analysis of free amino acids and protein hydrolysates (Alterman et al. Anal Biochem 335: 184-191, 2004). Linear responses between the amino acid concentration and the peak intensity ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 μM. Limit of quantitation varied from 0.03 μM for arginine to 3.7 μM for histidine and homocysteine. This method has one inherent limitation: the analysis of isomeric and isobaric amino acids. To solve this problem, a second protocol based on the use of MALDI TOF/TOF MS/MS for qualitative analysis of amino and organic acids was developed. This technique is capable of distinguishing isobaric and isomeric compounds (Gogichayeva et al. J Am Soc Mass Spectrom 18: 279-284, 2007). Both methods do not require amino acid derivatization or chromatographic separation, and the data acquisition time is decreased to several seconds for a single sample.

Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet

Ethoxyresorufin (EROD) and pentoxyresorufin (PROD) O-dealkylase activities, and contributions of the P450 cytochromes CYP1A1, CYP2B1 and 2, CYP2C6 and CYP2C12 to these metabolic activities, were evaluated in hepatic microsomes from ad libitum and calorie restricted female Fischer 344 rats across an age continuum from 1 to 26 months. The presence of CYP1A in microsome preparations was confirmed by western blot analysis. Uninduced levels of EROD and PROD peak in very young animals, decline to about 3 months of age, and do not exhibit an additional substantive decline after about 3 months of age. Monoclonal antibody (MAb) 1-7-1 (anti-CYP1A) strongly inhibited EROD activity in all microsome preparations, with the highest levels of inhibition in microsomes from 1- and 3-month-old AL animals. PROD activity in 1-month uninduced animals was apparently not attributable solely to CYP2B1 and 2, since it was inhibited by about 30% in both 1- and 26-month-old AL rats by an MAb specific for CYP2C12. However, in CR rats, CYP2C12 did not contribute to PROD activity. Approximately 40% of PROD activity in old AL rats and 20% of PROD activity in old CR rats was inhibited by an MAb specific for CYP2C6. These data indicate that long-term calorie restriction modulates either the expression or post-translational modification patterns of constitutive P450 isozymes in rats as they age, with P450 patterns in calorie restricted rats more closely resembling those found in young animals.

Hydroxylation of fatty acids by microsomal and reconstituted cytochrome P450 2B1

Understanding the mechanisms by which cytochrome(s) P450 (CYP) discriminate good from poor substrates, and orient them for highly regio‐ and stereoselective oxidation, has considerable intrinsic and practical importance. Here we present results of a study of fatty acid hydroxylation by CYP2B1 in a reconstituted system and in microsomes from phenobarbital‐pretreated rats. The results indicate that 2B1 prefers decanoic acid as the optimum fatty acid substrate (among C8–C16) and that it hydroxylates positions five or more methylene groups distant from the carboxylate carbon. That hydroxylation does not occur at carbon atoms closer to the carboxyl group than the C6 position suggests that these carbons may not reach the ferryl oxygen because the carboxyl group is anchored to a specific site at a fixed distance from the heme iron.