Search

alpha-enolase of Bacillus anthracis has recently been classified as an immunodominant antigen and a potent virulence factor determinant. alpha-enolase (2-phospho-d-glycerate hydrolase (EC 4.2.1.11), a key glycolytic metalloenzyme catalyzes the dehydration of d-(+)-2-phosphoglyceric acid to phosphoenolpyruvate. Interaction of surface bound alpha-enolase with plasminogen has been incriminated in tissue invasion for pathogenesis. B. anthracis alpha-enolase was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity that exhibited a K(m) of 3.3 mM for phosphoenolpyruvate and a V(max) of 0.506 microM min(- 1) mg(-1). B. anthracis whole cells and membrane vesicles probed with anti-enolase antibodies confirmed the surface localization of alpha-enolase. The specific interaction of alpha-enolase with human plasminogen (but not plasmin) evident from ELISA and the retardation in the native gel reinforced its role in plasminogen binding. Putative plasminogen receptors in B. anthracis other than enolase were also observed. This binding was found to be carboxypeptidase sensitive implicating the role of C-terminal lysine residues. The recombinant enolase displayed in vitro laminin binding, an important mammalian extracellular matrix protein. Plasminogen interaction conferred B. anthracis with a potential to in vitro degrade fibronectin and exhibit fibrinolytic phenotype. Therefore, by virtue of its interaction to host plasminogen and extracellular matrix proteins, alpha-enolase may contribute in augmenting the invasive potential of B. anthracis.

In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNA(CUU)Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an alpha-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-enolase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-alpha-enolase with a Kd 1.9 microM and approached saturation at 10 microM. Lysine-dependent plasminogen binding to r-alpha-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues epsilon-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activation rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.

Four forms (alpha alpha, beta beta, gamma gamma and alpha gamma) of enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) were purified from human brain and skeletal muscle. Each purified preparation showed a single band on SDS-polyacrylamide gel electrophoresis, with a relative mobility corresponding to a molecular weight of about 46 000 for alpha and gamma subunits, or 44 000 for the beta subunit. The four enzymes showed similar values of specific activity (about 80 units/mg), optimal pH (pH 6.8-6.9), and Km for 2-phosphoglycerate (about 3 X 10(-5) M). Amino acid analysis, peptide mapping analysis with a limited proteolysis, and immunochemical studies of the purified enzymes revealed that the three subunits, alpha, beta and gamma, are distinct proteins. Three hybrid enolases (alpha gamma, beta gamma and alpha beta) could be prepared from a mixture of two homodimeric enolases by dissociation and reassociation procedures, followed by isolation with ion-exchange column chromatography.

Human 14-3-2 protein, a nervous-system specific enolase (EC 4.2.1.11) isoenzyme, has been purified from human brain and a sensitive radioimmunoassay has been developed for its detection. A systematic survey of human organs has shown that immunoreactive nervous-system specific enolase is present in all human organs but at levels less than 3% of those found in human brain, with especially low levels in liver, kidney and skeletal muscle, and with the highest levels in adrenal and large intestine. In all organs immunoreactive nervous-system specific enolase occurs in two forms representing the heterodimer and homodimer forms of the enzyme, and in all tissues except brain the heterodimer predominates. The presence of nervous-system specific enolase in other organs is unlikely to be explicable by innervation alone since significant quantities are found in red blood cell haemolysates. Tissues which contain amine precursor uptake and decarboxylation cells, for which the protein has been claimed to be a specific molecular marker, do not contain significantly higher levels of immunoreactive nervous-system specific enolase than other tissues. Both the heterodimer and homodimer forms of the enolase appear to be expressed at low levels in all tissues.

1. The chromatography of rat brain enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) reveals three distinet components. One of these appears to be isoenzyme 1 (alpha alpha) but isoenzymes 2 (alpha beta) and 3 (beta beta) are absent. 2. The most acidic form of brain enolase was partially purified and an antiserum raised against it in the chicken. 3. a combination of chromatographic and immunological studies showed that a third type of subunit (gamma) is present in the brain giving rise to two further isoenzymes (alpha gamma and gamma gamma). 4. Developmental studies on the brain enzyme show an increase in total enolase activity from foetal life to maturity and a concurrent rise in the proportion of brain specific dimers. 5. It is therefore concluded that there are three genetic loci alpha, beta and gamma, coding for the enolase isoenzymes of rat tissues.

When a purified preparation of rat alpha gamma enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form alpha alpha and gamma gamma enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (alpha alpha and gamma gamma) of human brain enolase were purified from a crude preparation of the hybrid alpha gamma enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (alpha alpha) or a DEAE-Sephadex column (gamma gamma). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified alpha alpha and gamma gamma enolases revealed that the two subunits, alpha and gamma, are distinct proteins. The antisera to human alpha alpha or gamma gamma enolase cross-related with the respective form of rat enolase.

A human-rat hybrid form of enolase (beta gamma) was prepared by dissociation and reassociation of a mixture of human beta beta and rat gamma gamma enolases, followed by isolation of the hybrid form from the parental homodimeric enolases with DEAE-Sephadex column chromatography. The human-rat beta gamma enolase had a specific activity similar to those of human beta beta and rat gamma gamma enolases. The optimal pH, stability against heating, and Km for 2-phosphoglycerate of the hybrid enolase were also similar to those of the homodimeric enolases.

Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively.