Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features.

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.

The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis.

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.

Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli.

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.

HLA class I genotyping by cDNA sequence of a Vietnamese family expressing a weak B46 antigen.

Serological heterogeneity in HLA-B46 antigens has been described. Previous studies have identified B*4601 as the allele encoding the "strong" B46 antigen found in Chinese populations. Serological characterization of a Vietnamese family revealed a "weak" B46 antigen. Complementary DNA for the HLA-A, B and C alleles of three family members were cloned and the coding regions sequenced. The allele encoding the weak B46 antigen has the same coding sequence as B*4601, demonstrating that the antigenic differences are not due to polymorphism in the amino acid sequence of the HLA-B heavy chain. The recently described HLA-B*1525 and HLA-Cw*0403 alleles were also found to segregate in this Vietnamese family.

Peptides bound endogenously by HLA-Cw*0304 expressed in LCL 721.221 cells include a peptide derived from HLA-E.

The peptide-binding specificity of HLA-Cw*0304 was determined. Sequence analysis of endogenously-bound peptides isolated from Cw*0304 expressed by LCL 721.221 (221 for short) cells transfected with Cw*0304 cDNA revealed this class I allotype preferentially binds peptides possessing alanine at position 2 and leucine or methionine at the C-terminus. One peptide isolated from Cw*0304 expressed by 221 cells has sequence identity to residues 116-126 of HLA-E. Expression of HLA-E by 221 cells was confirmed by isolation of mRNA transcripts for HLA-E*0101 and detection of beta 2-microglobulin (beta 2-m)-associated HLA-E protein.

Subunit structure of vacuolar proton-pyrophosphatase as determined by radiation inactivation.

Vacuolar proton-pyrophosphatase (H(+)-PPase) of mung bean seedlings contains a single kind of polypeptide with a molecular mass of approx. 73 kDa. However, in this study, a molecular mass of approx. 140 kDa was obtained for the purified vacuolar H(+)-PPase by size-exclusion gel-filtration chromatography, suggesting that the solubilized form of this enzyme is a dimer. Radiation inactivation analysis of tonoplast vesicles yielded functional masses of 141.5 +/- 10.8 and 158.4 +/- 19.5 kDa for PP1 hydrolysis activity and its supported proton translocation respectively. These results confirmed the in situ dimeric structure of the membrane-bound H(+)-PPase of plant vacuoles. Further target-size analysis showed that the functional unit of purified vacuolar H(+)-PPase was 71.1 +/- 6.7 kDa, indicating that only one subunit of the purified dimeric complex would sufficiently display its enzymic reaction. Moreover, in the presence of valinomycin and KCl, the functional size of membrane-bound H(+)-PPase was decreased to approx. 63.4 +/- 6.3 kDa. A working model was proposed to elucidate the structure of native H(+)-PPase on vacuolar membrane as a functional dimer. Factors that would disturb the membrane, e.g. membrane solubilization and the addition of valinomycin and KCl, may induce an alteration in its enzyme structure, subsequently resulting in a different functional size.

Involvement of tyrosine residue in the inhibition of plant vacuolar H(+)-pyrophosphatase by tetranitromethane.

Plant vacuolar vesicles contain a novel H(+)-translocating pyrophosphatase (H(+)-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H(+)-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H(+)-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H(+)-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H(+)-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H(+)-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H(+)-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H(+)-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H(+)-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H(+)-PPase. Taken together, we suggest that vacuolar H(+)-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.

ATPase of Rhodospirillum rubrum requires three functional copies of beta subunit as determined by radiation inactivation analysis.

Radiation inactivation analysis yielded a functional unit of 170 +/- 26 kDa as beta subunit of ATPase was irradiated and then reconstituted to beta-depleted chromatophores of Rhodospirillum rubrum. A functional size of 132 +/- 17 kDa for the beta-depleted ATPase moiety involved in ATP hydrolysis reaction was also determined. When both purified beta subunit and beta-depleted chromatophore were irradiated separately, reconstituted, and then activity measured, the functional mass was 312 +/- 50 kDa. Our compelling evidence directly indicates that three functional copies of beta subunits were required for ATP hydrolysis.

Functional size analysis of F-ATPase from Escherichia coli by radiation inactivation.

A radiation inactivation technique was employed to determine the functional size of adenosine triphosphatase from Escherichia coli (EF0EF1-ATPase). Functional units of the membrane-bound and the soluble ATPases were estimated to be 300 +/- 39 and 295 +/- 32 kDa, respectively. The presence of the free radical scavenger dithiothreitol was crucial in measuring the radiation inactivation size of ATPase. When gramicidin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added, an increase in the functional mass of membrane-bound ATPase was observed. In contrast, valinomycin and KCl had hardly any effect on the functional size of ATPase. We also determined a functional unit of 355 +/- 33 kDa for proton translocation by a fluorescence quenching technique. A reconstitution study using irradiated coupling factor 1 (EF1)-depleted membrane revealed that the functional mass of the proton channel was 96 +/- 11 kDa. A similar functional size for ATP-Pi exchange and ATP hydrolysis implies that both reactions might utilize identical machinery. Furthermore, functional units of soluble EF1 for unisite (nonsteady state) and multisite (steady state) ATP hydrolysis were calculated as 200 +/- 32 and 298 +/- 32 kDa, respectively. A working hypothesis was proposed from radiation inactivation analysis to elucidate the structure and mechanism of F1-ATPase.

Functional size of the thylakoid phosphatases determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of phosphatases from thylakoid membrane. The enzymatic activities of phosphatases decayed in a simple function with the increase of radiation dosage. D37 values of 18.8 +/- 2.4-14.1 +/- 1.5 Mrad were obtained, using phosphoserine, phosphothreonine, p-nitrophenol phosphate, and phospho-histone V-S, respectively, as substrates. The molecular masses of 48.2 +/- 6.3-61 +/- 5.7 kDa were yielded by target theory analysis. We thus speculate that the thylakoid alkaline phosphatase is probably a monomer while acid phosphatase is functionally a dimer in situ.

Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP.

The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.