Pyruvate analysis

The enantiomeric excess (ee) of the hydrogenated products was determined either by polarimetry, GLC equipped with a chiral column or H-NMR with a chiral shift reagent. Methyl lactate and methyl 3-hydroxybutanoate, obtained from 1 and 2, respectively, were analized polarimetry using a Perkin-Elmer 243B instrument. The reference values of [a]o(neat) were +8.4° for (R)-methyl pyruvate and -22.95° for methyl 3-hydroxybutcinoate. Before GLC analysis, i-butyl 5-hydroxyhexanoate, methyl 5-hydroxyhexanoate, and n-butyl 5-hydroxyhexanoate, obtained from 1, 5, and 6, respectively, were converted to the pentanoyl esters, methyl 3-hydroxybutanoate was converted to the acetyl ester, and methyl 4-methyl-3-hydroxybutanoate obtained from 2 was converted the ester of (+)-a-methyl-a-(trifluoromethyl)phenyl acetic acid (MTPA). 

The conformational analysis of methyl pyruvate shows that it can have two conformers. In the second conformer the two carbonyls are in syn position. The anti-syn conformational change requires 3 kcal. The [CDqIq qJ - methyl pyruvate ] complex ((R) form) was also calculated and shown in Figure 8. In the above complex the "directionality" of the lone pair of electrons of the quinuclidine nitrogen is advantageous for interactions with both the keto and the ester carbonyl groups. 

B3. Baronciani, L., and Beutler, E., Analysis of pyruvate kinase-deficiency mutations that produce nonspherocytic hemolytic anemia. Proc. Natl. Acad. Sci. U.S.A. 90,4324-4327 (1993). 

K6. Kanno, H., Fujii, H., and Miwa, S., Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells. Biochem. Biophys. Res. Commun. 188, 516-523 (1992). 

K11. Kanno, H Fujii, H., and Miwa, S Molecular heterogeneity of pyruvate kinase deficiency identified by single strand conformational polymorphism (SSCP) analysis. Blood 84 (Suppl. 1), 13a... 

Composition and Structure. Chemical analysis of the polymer from our first strain (NCIB 11592) indicated that it was a polysaccharide containing the sugars, glucose and galactose, and the carboxylic acids - - succinic and pyruvic - - in the approximate ratios 7 1 1 1. 

The homolytic acylation of protonated heteroaromatic bases is, as with alkylation, characterized by high selectivity. Only the positions a and y to the heterocyclic nitrogen are attacked. Attack in the position or in the benzene ring of polynuclear heteroaromatics has never been observed, even after careful GLC analysis of the reaction products. Quinoline is attacked only in positions 2 and 4 the ratio 4-acyl- to 2-acylquinoline was 1.3 with the acetyl radical from acetaldehyde, 1.7 with the acetyl radical from pyruvic acid, and 2.8 with the benzoyl radical from benzaldehyde. 

Amplification of the sensitivity of substrate or co-en me recycling is especially efficient in thermometric analysis since all the reactions involved frequently contribute to increasing the overall temperature change. One case in point is the determination of lactate or pyruvate by substrate recycling using co-immobilized lactate oxidase and lactate dehydrogenase [160]. 

Figure 11.9 Schematic view of the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes, (i) Freeze-thaw (x 7) solution containing t-RNAP , poly(U), Phe, ATP, GTP, Mg(OAc)2, NH4CI, spermine, spermidine, phos-phoenolpyruvate. (ii) Soution containing pyruvate kinase, 100 000 g supernatant enzymes, 308 and SOS ribosomal subunits, (iii) 1. Free-thaw (x3) 2. Brief extrusion 3. Addition of EDTA (final concentration = 35 mM. (iv) Withdrawl of aliquots at indicated time and cold TCA precipitation. Analysis of the radioactivity remaining on the glass filter by p-scintillation counting. (Modified from Oberholzer et al, 1999.)...

Fig. 8 SDS-PAGE (a) and Western blot (b) analysis of the purified hydrogenosomal fractions isolated from the metronidazole-susceptible T. vaginalis strain TV 10-02 (P) and its metronidazole-resistant derivatives MR-3, MR-5, MR-30, MR-50, and MR-100 displaying the aerobic (3), early anaerobic (5), advanced anaerobic (30, 50), and fully developed anaerobic resistance (100) to metronidazole. Numbers in the designation of MR strains indicate the concentrations of metronidazole in ixg/ml at which the organisms multiply in culture. About 10 pg protein was loaded per line. PFOR pyruvate ferredoxin oxidoreduc-tase, a-STK a subunit of succinate thiokinase (hydrogenosomal enzyme not involved in metronidazole resistance used as control), Fdx ferredoxin. From Rasoloson et al. (2002) by courtesy of the Society of General Microbiology...

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