The conformations of the adenosine moiety of MgADP and MgATP bound to rabbit muscle creatine kinase were investigated by two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The effects arising from adventitious binding of the ligands to the enzyme on the measurements were delineated. It was shown that, with sample protocols typically used thus far with the TRNOE method (enzyme, approximately 1 mM; ligand, approximately 10 mM), the TRNOESY pattern for the nucleotides with creatine kinase is similar to that with gamma-globulin and bovine serum albumin, which do not have specific nucleotide binding site(s). Measurements of NOE between the H1'-H2' proton pair as a function of ligand concentrations with the enzyme-ligand ratio kept constant at 1:10 showed that, for ligand concentrations over about 3-4 mM, weak nonspecific binding makes a significant contribution to the observed NOE. Thus the NOE values relevant for the determination of the nucleotide conformation at the active site were measured at nucleotide concentrations of about 1.5 mM. The TRNOE buildup curves for all the ligand-proton pairs were analyzed using a complete relaxation matrix approach. The interproton distances derived from the NOE's were then used as constraints in elucidating the ligand structure by using the program CHARMm. The NOE-determined structures of both MgADP and MgATP bound to creatine kinase correspond to an anti conformation with the glycosidic angle (O'4-C'1-N9-C8) chi = 51 +/- 5 degrees. The ribose pucker nominally representative of these data is a O4'T with a phase angle of pseudorotation (p) of 70.5 degrees.

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

The enolase protein of the human malarial parasite Plasmodium falciparum has recently been characterized. Apart from its glycolytic function, enolase has also been shown to possess antigenic properties and to be present on the cell wall of certain invasive organisms, such as Candida albicans. In order to assess whether enolase of P. falciparum is also antigenic, sera from residents of a region of Eastern India where malaria is endemic were tested against the recombinant P. falciparum enolase (r-Pfen) protein. About 96% of immune adult sera samples reacted with r-Pfen over and above the seronegative controls. Rabbit anti-r-Pfen antibodies inhibited the growth of in vitro cultures of P. falciparum. Mice immunized with r-Pfen showed protection against a challenge with the 17XL lethal strain of the mouse malarial parasite Plasmodium yoelii. The antibodies raised against r-Pfen were specific for Plasmodium and did not react to the host tissues. Immunofluorescence as well as electron microscopic examinations revealed localization of the enolase protein on the merozoite cell surface. These observations establish malaria enolase to be a potential protective antigen.Malaria continues to be a life-threatening infectious disease in the tropical world. Despite tremendous efforts to control the malaria epidemic, current prophylaxis and drug treatments are proving insufficient. The extensive spreading of drug-resistant Plasmodium strains as well as insecticide-resistant mosquitoes makes it urgent to develop an effective malaria vaccine. Long years of antigen identification and characterization have yielded many potential vaccine candidates, but developing an effective malaria vaccine has remained an incredibly difficult challenge (17). It has been observed that immunity to the disease develops gradually, after many attacks and over many years, in adults living in areas where malaria is endemic (2). The successful passive transfer of this immunity by injecting antibodies from malaria-immune persons to children susceptible to malaria has demonstrated that antibodies alone can trigger protection (5, 9, 58). These experiments have worked across geographical borders, as immunoglobulin G (IgG) from malaria-immune West Africans have cured East Africans as well as Thai malaria patients (5). The nature of this immunity is poorly understood at the molecular level. However, attempts have been made to identify antigens, the humoral response against which leads to protection. Seroepidemiological studies have identified several specific malarial blood-stage antigens including ring-infected erythrocyte surface antigen (10), apical membrane antigen (53), and PfP0, a conserved ribosomal protein (7,18,24), as protective antigens.Enolase has been reported to be present on the cell surface of several organisms (38). It is also considered to be a major immunostimulatory protein in the case of visceral leishmaniasis (19). Enolase has been demonstrated to play a protective role in Candida albicans infection (31, 41, 55). Recently, it has also been ...

BackgroundIn an earlier study, it was observed that the vaccination with Plasmodium falciparum enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.MethodsSub-cellular localization of P. falciparum enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.ResultsEnolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.ConclusionDiverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His 6 -tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities ( 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean ± SD mass of 51396 ± 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of 30 UAEmg )1 and K m2PGA ¼ 0.041 ± 0.004 mM. TheMichaelis constant for the reverse reaction (K mPEP ) is 0.25 ± 0.03 mM. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na + , whereas K + has a slight activating effect. The cofactor Mg 2+ has an apparent activation constant of 0.18 ± 0.02 mM. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein.