UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.

Production of black tea [BT] results in biotransformation of catechins of green tea [GT] to theaflavins and thearubigins. BT was found to be more efficient than GT and its individual catechin constituents in proportionate amounts in abrogating production of NO and O−2 in activated murine peritoneal macrophages. In a reconstitution system of BT that is free of all catechins, stepwise addition of catechins showed that though all the constituents contributed to the overall effect of BT, theaflavin was the most powerful in abrogating NO production. RT-PCR analysis also showed theaflavin to be the most important constituent in down-regulating synthesis of iNOS. Clearly, BT containing theaflavin is an excellent chemopreventor against reactive oxygen and nitrogen species.

Leishmania donovani is a protozoal pathogen that belongs to the kinetoplastida order. Unlike in other eucaryotic systems, the first three enzymes of the de novo pyrimidine biosynthetic pathway are not components of a multifunctional protein system. The three enzyme activities in the crude extract were separated on a Sephacryl S-200 column. Aspartate carbamoyltransferase (EC 2.1.3.2) has been purified to apparent homogeniety. The enzyme has an approximate molecular weight of 135,000 and seems to be a tetramer of equivalent subunits of molecular weight 35,000. The enzyme shows strictly hyperbolic kinetics with both the substrates under a variety of conditions and is not inhibited by nucleotide phosphates. K,,, for carbamyl phosphate is 3.1 X M and for aspartate is 7.6 X M. Apparently, the enzyme has no regulatory role in pyrimidine biosynthesis. N-(Phosphonoacety1)-L-aspartic acid is a powerful competitive inhibitor (Ki = 5 X M) for this enzyme with carbamyl phosphate as substrate. This inhibitor completely inhibits the growth of the vector form of organism at 60 WM and significantly affects the growth of the pathogenic form in a macrophage assay system, The potency of the inhibitor is comparable with allopurinol which is undergoing human clinical trial as an antileishmanial drug.

Hamycin, a polyene antibiotic, now in extensive use in the treatment of candidiasis and otomycosis, is found to be remarkably effective in killing Leishmania donovani promastigotes in a liquid medium at a concentration of 0.2 microgram/ml. The glucose stimulated respiration and the uptake of 2-deoxy-D[U-14C]-glucose was inhibited in cells treated with the drug at a growth inhibitory concentration. An immediate release of isotopic glucose from preloaded cells could be demonstrated after exposure to hamycin. All the above effects could be effectively prevented in the presence of ergosterol. The primary site of action of hamycin on L. donovani promastigote cells appears to be membrane sterols that result in the loss of the permeability barrier to small metabolites. The lower minimum inhibitory concentration of hamycin compared to other established drugs warrants further study in the context of increasing reports of clinical resistance to pentavalent antimonials.

Acivicin, a chlorinated amino acid antibiotic, is found to be remarkably effective in killing both the vector and the host form of the parasitic protozoa, Leishmania donovani, the causative agent for visceral leishmaniasis or Kala-azar. The ED50 (50 nM) for the pathogenic amastigote form in in vitro screening system is significantly lower than the reported values for other drugs under trial. The drug irreversibly inactivates both in vitro and in vivo carbamyl phosphate synthetase II, the first enzyme of the pyrimidine biosynthetic pathway. The irreversible inactivation of this sensitive target enzyme and lack of effective reversal by glutamine makes acivicin a preferred candidate for potential chemotherapy against increasing number of Kala-azar cases that are reported to be unresponsive to pentavalent antimonials.

The growth of Leishmania donovani promastigotes in a liquid medium was completely inhibited by amphotericin B at a concentration of 0.3 μm ml−1 (0.3 μM). Continuous release of small molecules that absorb at 260 nm and 280 nm was observed after contact with the drug. Uptake of [U-14C]glucose was inhibited in cells treated with the drug. An immediate release of isotopic glucose and its metabolites from preloaded cells could be demonstrated after incubation with amphotericin B (0.4 μM). Inhibition of respiration by the drug was a comparatively slower process. All the above effects could be effectively prevented in the presence of either cholesterol or ergosterol. The primary site of action of amphotericin B on L. donovani promastigote cells appears to be membrane sterols that result in a loss of the permeability barrier to small metabolites. An interesting biochemical similarity, thus, emerges between flagellated protozoa and fungi.

Uridine 5′-diphosphate glucose 4-epimerase (EC 5.1.3.2) from Ehrlich ascites carcinoma cells was purified to apparent homogeneity using conventional procedures and NAD-hexane-agarose affinity chromatography. The protein had a molecular weight of 96,000. The ascites enzyme had an absolute requirement for exogenously added NAD (10 ΜM) for stability. This appears to be a unique feature of ascites epimerase since epimerase from other mammalian sources did not exhibit such a dependence. Exogenously added NAD was also needed for catalysis with an apparentK m value of 2.5 ΜM. NADH was a very potent competitive inhibitor (K i = 0.11 ΜM with respect to NAD) of the enzyme activity at pH values close to intracellular pH. The dependence of the enzyme on NAD for stability and its inhibition by NADH may have some potential significance in tumor metabolism

UDPglucose 4-epimerase (EC 5.1.3.2) from Saccharomyces fragilis is a holoenzyme containing 1 mol NAD per mol dimeric protein. The enzyme can be dissociated with p-chloromercuribenzoate and can be reconstituted in the presence of 2-mercaptoethanol and exogenous NAD. Using Cibacron blue F3GA in this reconstituting system, competition between NAD and the dye for the pyridine nucleotide-binding site could be demonstrated. Inactive holoenzyme containing Cibacron blue can also be obtained under these conditions. These data suggest the possible presence of elements of a dinucleotide fold in this enzyme.

The allosteric kinetics exhibited by UDP glucose 4-epimerase from Saccharomyces fragilis changes over to a normal hyperbolic kinetics when the enzyme is heated at 41° for 2 mins. The native enzyme is completely insensitive to inhibition by UMP in the allosteric region. The desensitized enzyme is however, strongly inhibited by UMP at this low concentrations. Apparently, desensitization by heat converts the enzyme to its ultimate catalytic form.

This chapter presents a table that consist of enzymic synthesis of various sugar nucleotides. Individual enzymes are numbered according to the enzyme commission numbers based on the enzyme nomenclature recommendation of the International Union of Biochemistry. It is noted that organic synthesis of most sugar nucleotides can be readily accomplished. The sugar nucleotides are divided into 11 groups as listed below. Aldopentoses; aldohexoses and aldoheptoses; alditols; uronic acids; deoxy sugars; branched carbohydrates; amino sugars; aminouronic acids; keto acids; muramic acid and muramyl peptides; and oligosaccharides and sulfate-substituted carbohydrates. Sugar nucleotides with sulfate-substituted carbohydrates or with oligomeric carbohydrate chains are listed although no biological role has been established for them.

This chapter presents a table that consist of enzymic synthesis of various sugar nucleotides. Individual enzymes are numbered according to the enzyme commission numbers based on the enzyme nomenclature recommendation of the International Union of Biochemistry. It is noted that organic synthesis of most sugar nucleotides can be readily accomplished. The sugar nucleotides are divided into 11 groups as listed below. Aldopentoses; aldohexoses and aldoheptoses; alditols; uronic acids; deoxy sugars; branched carbohydrates; amino sugars; aminouronic acids; keto acids; muramic acid and muramyl peptides; and oligosaccharides and sulfate-substituted carbohydrates. Sugar nucleotides with sulfate-substituted carbohydrates or with oligomeric carbohydrate chains are listed although no biological role has been established for them.

Long-chain fatty acids are synthesized from citrate in an undialyzed soluble enzyme system from pigeon liver at a rate comparable to that observed when acetate is used as a precursor. The first enzymic step for citrate incorporation is probably its breakdown to acetyl-CoA and oxaloacetate catalyzed by the citrate-cleavage enzyme. Experiments with variously labeled citrate show good incorporation of the acetyl portion of citrate into fatty acids, and a poor incorporation of the oxaloacetyl portion into fatty acids. The incorporation of the oxaloacetyl portion of citrate as well as the acetyl portion has been shown to be avidin sensitive. The enzymic sequence from the oxaloacetyl portion remains uncertain.

While the detrimental effects of binge drinking are well recognized, low-to-moderate alcohol consumption may be beneficial to health, although the underlying mechanism(s) remains elusive. In this opinion article, we will examine the effects of low dose alcohol consumption from the perspective of epigenetic modulation. Biochemically, alcohol is metabolized into acetate and subsequently to acetyl-coA, which can modulate histone acetylation levels. While elevated levels of acetyl-CoA are detrimental for longevity, we argue that diminished acetyl-CoA also negatively affects fatty acid biosynthesis and histone acetylation, which play a critical role in gene expression and, ultimately, health span. Since mitochondrial function and glucose metabolism, which provide the main source of nucleocytoplasmic acetyl-CoA, are compromised with age, alcohol-derived acetate could be an alternative source of acetyl-CoA to compensate. Hence, the health benefits of low ethanol consumption may be more pronounced after midlife, since mitochondrial function and/or glucose metabolism are diminished in this phase of the life course. Indeed, various clinical alcohol consumption studies concur with this notion, and have shown that a low dose of regular alcohol intake after midlife brings about various health and survival benefits. The requirement for regular alcohol intake may also reflect the transient nature of ethanol-induced histone acetylation. Conversely, ethanol may also stimulate carcinogenesis by inhibiting DNA methylation, as it was shown to reduce various pathways leading to DNA and histone methylation. However, unlike acetylation, where ethanol directly increases the substrate for acetylation, this effect was only observed in the high alcohol exposure cohort. While alcohol-derived acetate may be beneficial for health after midlife, various detrimental effects of alcohol consumption remain, and hence, we do not advocate excessive drinking to increase acetate. This opinion article establishes a possible role of ethanol-derived acetate in achieving homeostasis and sustaining an organism's health span.