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Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that l-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5'-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N- and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

After exposure to [U-(13)C3]glycerol, the liver produces primarily [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other (13)C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex (13)C-labeling patterns in exported glucose. Here, we investigated (13)C labeling in plasma glucose in rats given [U-(13)C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a (13)C-(13)C bond within the trioses contributing to glucose, the appearance of [1,2-(13)C2]-, [2,3-(13)C2]-, [5,6-(13)C2]-, and [4,5-(13)C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-(13)C2]glucose/[2,3-(13)C2]glucose was significantly greater than [5,6-(13)C2]glucose/[4,5-(13)C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by (13)C distribution in blood glucose after [U-(13)C3]glycerol administration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Accumulating genome sequences have revealed the existence of a large number of conserved hypothetical proteins. Characterization of these proteins is considered essential in the elucidation of intracellular biological pathways. Our previous transcriptomic analysis suggested that, in Thermus thermophilus HB8, loss of an oxidized DNA-repairing activity leads to the up-regulation of a function-unknown gene, tthb071, which is conserved in a wide range of bacteria. Interestingly, the tthb071 gene product, TTHB071, showed a significant primary structure similarity to apurinic/apyrimidinic (AP) endonucleases, which are required for the repair of oxidized DNA. In the present study, we observed that disruption of tthb071 increases the H(2)O(2) sensitivity in T. thermophilus HB8, suggesting the involvement of tthb071 in a protection mechanism against oxidative stress. However, purified TTHB071 exhibited no AP endonuclease or DNA-binding activities, indicating that TTHB071 plays no major role in repairing oxidative DNA damage. Then we determined the three-dimensional structure of TTHB071 complexed with zinc ions by x-ray crystallography. In addition to the overall structural similarity, the zinc-binding fashion was almost identical to that of the phosphatase active site of an AP endonuclease, implying that TTHB071 possesses a phosphatase activity. Based on the structural information around the zinc-binding site, we investigated the binding of TTHB071 to 14 different compounds. As a result, TTHB071 favorably bound FMN and pyridoxal phosphate in a zinc ion-mediated manner. Our results suggest that TTHB071 protects the cell from oxidative stress, through controlling the metabolism of FMN, pyridoxal phosphate, or an analogous compound.

The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor compartments by separating the entire photoreceptor layer of a flat-mounted frozen retina into a series of thin tangential cryosections and analyzing protein compositions of each section by label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 protein database entries, we generated a quantitative proteomic database (a "map") correlating the distribution profiles of identified proteins with the profiles of marker proteins representing individual compartments of photoreceptors and adjacent cells. We evaluated the applicability of several common peptide-to-protein quantification algorithms in the context of our database and found that the highest reliability was obtained by summing the intensities of all peptides representing a given protein, using at least the 5-6 most intense peptides when applicable. We used this proteome map to investigate the distribution of glycolytic enzymes, critical in fulfilling the extremely high metabolic demands of photoreceptor cells, and obtained two major findings. First, unlike the majority of neurons rich in hexokinase I, but similar to other highly metabolically active cells, photoreceptors express hexokinase II. Hexokinase II has a very high catalytic activity when associated with mitochondria, and indeed we found it colocalized with mitochondria in photoreceptors. Second, photoreceptors contain very little triosephosphate isomerase, an enzyme converting dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This may serve as a functional adaptation because dihydroxyacetone phosphate is a major precursor in phospholipid biosynthesis, a process particularly active in photoreceptors because of the constant renewal of their light-sensitive membrane disc stacks. Overall, our approach for proteomic profiling of very small tissue amounts at a resolution of a few microns, combining cryosectioning and liquid chromatography-tandem MS, can be applied for quantitative investigation of proteomes where spatial resolution is paramount.

In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the "inositol-less death" phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis.

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.

We reported previously that cofilin, an actin-binding protein, interacts with Na,K-ATPase and enhances its activity (Lee, K., Jung, J., Kim, M., and Guidotti, G. (2001) Biochem. J. 353, 377-385). To understand the nature of this interaction and the role of cofilin in the regulation of Na,K-ATPase activity, we searched for cofilin-binding proteins in the rat skeletal muscle cDNA library using the yeast two-hybrid system. Several cDNA clones were isolated, some of which coded for triose-phosphate isomerase, a glycolytic enzyme. The interaction of cofilin with triose-phosphate isomerase as well as Na,K-ATPase was confirmed by immunoprecipitation and confocal microscopy in HeLa cells. Cofilin was translocated to the plasma membrane along with triose-phosphate isomerase by the Rho activator lysophosphatidic acid but not by the p160 Rho-associated kinase inhibitor Y-27632, suggesting that the phosphorylated form of cofilin bound to TPI interacts with Na,K-ATPase. Ouabain-sensitive (86)Rb(+) uptake showed that Na,K-ATPase activity was increased by the overexpression of cofilin and lysophosphatidic acid treatment, but not by the overexpression of mutant cofilin S3A and Y-27632 treatment. Pretreatment with the glycolytic inhibitor iodoacetic acid caused a remarkable reduction of Na,K-ATPase activity, whereas pretreatment with the oxidative inhibitor carbonyl cyanide m-chlorophenylhydrazone caused no detectable changes, suggesting that the phosphorylated cofilin is involved in feeding glycolytic fuel for Na,K-ATPase activity. These findings provide a novel molecular mechanism for the regulation of Na,K-ATPase activity and for the nature of the functional coupling of cellular energy transduction.

Macromolecular crowding, in principle, should affect any reaction that is accompanied by significant reduction in excluded volume. Here we have examined the influence of crowding on reverse proteolysis. We show that proteosynthesis of a polypeptide product with an interacting folding motif such as coiled coil is facilitated in crowded media as a consequence of the volume exclusion effect. Further, we demonstrate that crowding could also effect the conversion of a noncovalent protein complex (fragment complementing protein) obtained by limited proteolysis to the native covalent form, but only if the formation of the native protein results in large compaction leading to a substantial volume exclusion effect. Subtilisin-catalyzed reformation of native triosephosphate isomerase (TIM) from multiple fragments is facilitated by crowding. However, a single nick in ribonuclease S (RNase S) could not be ligated under similar conditions. The failure of generation of RNase A from RNase S is consistent with the fact that the crystal structure of the two forms are almost superimposable, and hence no significant difference of volume exclusion exists between reactant (RNase S) and product (RNase A). In contrast, considerable compaction, and consequently large reduction in excluded volume, is attained through the assembly of a TIM barrel structure. Taken together, these results have implications for both in vitro as well as in vivo polypeptide assemblage by reverse proteolysis.

We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3- and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase Calpha, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.

The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-A resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15 degrees C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25 degrees C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The Td values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54 degrees C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.

For a given biochemical transformation, such as the fermentation reaction, the redistribution coefficients, which relate the natural site-specific isotope contents in end products to those of their precursors, are a source of mechanistic information. These coefficients characterize the traceability of specific hydrogens in the products (ethanol and water) to their parent hydrogens in the starting materials (glucose and water). In conditions of complete transformation, they also enable intermolecular exchanges with the water medium to be estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6' of glucose are strongly connected to the methyl site I of ethanol obtained by fermentation by Saccharomyces cerevisiae. However, whereas hydrogens 6 and 6' are transferred to a great extent, transfer is only partial for hydrogen 2, and it is even less for hydrogen 1. Because the two moieties of glucose corresponding to carbons 1-2-3 and 4-5-6 are scrambled by the aldolase and triosephosphate isomerase reactions, additional exchange of hydrogens at positions 1 and 2 must have occurred before these steps. The value of the coefficient that relates site 2 of glucose to site I of ethanol in particular can be used to quantify the contribution of intermolecular exchange occurring in the course of the transfer from site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate mediated by phosphoglucoisomerase. The average hydrogen isotope effects associated with the transfer of hydrogen from the water pool to the methyl or methylene site of ethanol are estimated. In contrast to conventional experiments carried out in strongly deuterium-enriched media where metabolic switching may occur, the NMR investigation of site-specific natural isotope fractionation, which operates at tracer isotopic abundance, faithfully describes the unperturbed metabolic pathways.

The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH3 (1), C6H5 (2), C6H4Cl (3), or C6H4Br (4). For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction. However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93. In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold. These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site. Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I (100-111) and Pseudomonas putida Glx I (93-105), that shows significant sequence homology to the "catalytic loop" of chicken muscle triosephosphate isomerase (TIM) (165-176). The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P. putida Glx I. Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

Others have concluded that beta-fructose 1, 6-bisphosphate is a substrate for muscle aldolase on the basis of rapid kinetic measurements. In view of new data showing excellent aldol cleavage of an analog of the keto form and a very high rate of spontaneous ring opening, Midelfort et al. (Midelfort, C. F., Gupta, R., and Rose, I.A. (1976) Biochemistry 15, 2178-2185) have suggested that the beta form may be used only after spontaneous conversion to the keto form in solution, followed by reaction of the keto form with the aldolase. In order to determine whether beta-fructose-1, 6-P2 is itself a substrate a steady state approach was devised in which the beta form is specifically produced by phosphofructokinase for use by the aldolase present. Since aldolase binds very tightly to bisphosphates similar in size to beta-fructose-1, 6-P2, it was expected that if a spontaneous ring opening were essential for catalysis, it would be possible to decrease the rate of production of triose-Ps by using very high concentrations of aldolase. If the beta form were itself a substrate, the rate would reach a constant value with increasing aldolase, limited by the phosphofructokinase rate. It was found that under conditions where only approximately 2 per cent of the total fructose-1, 6-P2 present in the steady state would be free, the turnover of the complexed fructose-1, 6-P2 was about 20-fold greater than that in which spontaneous ring opening is a required step. Using similar methods, the turnover of enzyme-bound glyceraldehyde-P and dihydroxyacetone-P were determined. It was concluded that at saturation both the beta and acyclic forms have about the same rates as substrates.

While the equilibrium assumption and the validity of using total measured concentrations for near equilibrium indicator reactions have been widely tested in liver, these have not been systematically evaluated in skeletal muscle. Vascularly isolated dog gracilis muscles were stimulated via the nerve at 4 Hz, and tissue was sampled by quick freezing at rest and after 10, 15, 30, 60, and 180 s of stimulation or after stimulation in the presence of glycolytic blockade by iodoacetate. Phosphocreatine, creatine, and several glycolytic intermediates were measured in tissue extracts. The in vivo mass action ratios for triosephosphate isomerase and aldolase were evaluated relative to substrate concentrations and compared with equilibrium constants determined in vitro. Although there was evidence of substrate binding at low substrate levels for the triosephosphate isomerase reaction, the in vivo mass action ratios for both reactions stabilized at a constant value at moderate substrate levels and in glycolytically blocked muscles. It was concluded that both enzymes are in apparent equilibrium in vivo, but the equilibrium constants are lower than those determined in vitro. The mass action ratios of the combined creatine kinase, lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase reactions were determined for resting muscles. These reactions are also at equilibrium and the equilibrium constants are consistent with in vitro values.