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Biomedical subjects

F Strasser

Publications and source records attributed to F Strasser.

18 recordsLinked to original sources
PubMed2001

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase.

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.

Acetates↗
PubMed2000

Giant cell arteritis "causing" AA-amyloidosis with rapid renal failure.

Giant cell arteritis (GCA) is a systemic vasculitic disease, which in very rare cases causes inflammatory complications such as secondary amyloidosis. We describe a well-documented case, with a clinically mild course, of biopsyproven giant cell arteritis as the only apparent cause of systemic AA-Amyloidosis. The deterioration in renal function due to amyloid deposition occurred rapidly and only a few months after manifestation of giant cell arteritis and was not reversible by steroid treatment. The renal arteries were normal and there was no glomerulonephritis due to giant cell arteritis. This unique case demonstrates that giant cell arteritis with a mild clinical course is closely associated with early-onset severe secondary amyloidosis.

Acute Kidney Injury↗
PubMed1998

Activation of horse liver alcohol dehydrogenase upon substitution of tryptophan 314 at the dimer interface.

Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 buried in the interface between the subunits of the dimer. We studied the contributions of the tryptophans to fluorescence and catalytic dynamics by substituting Trp-314 with a leucine residue and making two compensatory mutations that were required to obtain a stable protein, leading to the triple mutant M303F-L308I-W314L enzyme. The substitutions increased by two- to sixfold the turnover numbers for ethanol oxidation, acetaldehyde reduction, and the dissociation constants of the coenzymes. The rate of the exponential burst phase for the transient oxidation of ethanol increased slightly, but the rate of dissociation of the enzyme-NADH complex still limited turnover of ethanol, as for wild-type enzyme. The three substitutions at the dimer interface apparently activate the enzyme by allowing more rapid conformational changes that accompany coenzyme binding, probably due to movement of the loop containing residues 293 to 298. The emission spectrum of M303F-L308I-W314L enzyme, which contains Trp-15, was redshifted compared to wild-type enzyme. Time-resolved fluorescence measurements with the triple mutant show that the decay of Trp-15 is dominated by a approximately 7-ns component. In the mutant enzyme with Trp-15 substituted with phenylalanine, the decay of Trp-314 is dominated by a approximately 4-ns component. Solute quenching data for wild-type enzyme and the mutants show that only Trp-15 is exposed to iodide and acrylamide, whereas Trp-314 is inaccessible. The luminescence properties of the tryptophan residues in the mutated enzymes are consistent with conclusions from studies of the wild-type enzyme [M. R. Eftink, 1992, Adv. Biophys. Chem. 2, 81-114].

Alcohol Dehydrogenase↗
PubMed1998

Inhibition of myo-inositol monophosphatase isoforms by aromatic phosphonates.

alpha-Hydroxyphosphonates are moderately potent (Ki = 6-600 microM) inhibitors of the enzyme myo-inositol monophosphatase (McLeod et al., Med. Chem. Res. 1992, 2, 96). Hydroxy-[4-(5,6,7,8-tetrahydronaphtyl-1-oxy)phenyl]methyl phosphonate (3) was resynthesized and its inhibitory potency towards the recombinant bovine brain enzyme confirmed (Ki = 20 microM). Similar aromatic difluoro-, keto-, and ketodifluorophosphonates (5, 7, 9) were inactive. Compound 3 was 15-fold less active on the human as compared to the bovine enzyme. Molecular modeling suggested that the hydrophobic part of the inhibitor interacts with amino acid side chains that are located at the interface between the enzyme subunits in an area (amino acids 175-185) with low similarity between the two isozymes. Phe-183 in the human enzyme was replaced with leucine, the corresponding residue in the bovine isoform. The three isozymes (human wild-type, bovine wild-type and human F183L) had similar kinetic properties, except that the bovine enzyme was less effectively inhibited by high concentrations of the activator Mg2+. The F183L mutant enzyme had a twofold increased affinity for compound 3 as compared to the human wild-type form. We conclude that residue 183 contributes to the binding of aromatic hydroxyphosphonates to IMPase, but it is not the only determining factor for inhibitor specificity with respect to different isozymes.

5'-Nucleotidase↗
PubMed1997

Elevated serum macrophage inhibitory factor-related protein (MRP) 8/14 levels in advanced HIV infection and during disease exacerbation.

UNLABELLED: To assess the value of MRP 8, MRP 14, and MRP 8/14 serum concentrations as markers of disease progression in HIV infection and as markers of intercurrent infections. DESIGN: We measured MRP 8, MRP 14, and MRP 8/14 serum concentrations in 184 HIV-infected patients in various stages of disease with or without disease exacerbation and in 50 healthy control subjects. In clinically stable HIV-infection correlations of MRP levels with stage of HIV disease, CD4 counts, p24 antigen, and beta-2 microglobulin levels were studied. In patients with intercurrent illnesses, correlations of MRP levels with type of disease exacerbation and with CRP were calculated and compared with those found in stable HIV infection. RESULTS: MRP 8/14 levels were significantly elevated and MRP 8 levels slightly decreased in stable HIV infection compared with HIV-negative controls. The CD4 cell count and MRP 8/14 levels correlated significantly in patients with AIDS. Despite higher values of MRP 8/14 during advanced disease, these were not significant predictors of progression to death. In patients with acute infections, MRP 8/14 levels were significantly elevated, compared with patients with illnesses of noninfectious origin. Levels of MRP 8/14 associated with acute infections were significantly higher in patients with AIDS than in patients during earlier stages of HIV infection. CONCLUSIONS: Both stable HIV infection and advanced immunedeficiency are associated with an elevation of the MRP 8/14 complex and probably with a decline of MRP 8 serum levels. MRP 8/14 is preserved as a marker of acute infection in immunecompromised patients.

Acute Disease↗
PubMed1996

The contribution of lysine-36 to catalysis by human myo-inositol monophosphatase.

The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was completely inactivated by amidination with ethyl acetimidate or reductive methylation with formaldehyde and cyanoborohydride. Activity was retained when the active site was protected with Mg2+, Li+, and D,L-myo-inositol 1-phosphate. Using radiolabeling, peptide mapping, and sequence analysis, Lys-36 was shown to be the protected residue, which is responsible for inactivation. Replacing Lys-36 with glutamine produced a mutant protein, K36Q, with similar affinities for the substrate and the activator Mg2+, but a 50-fold lower turnover number as compared to the wild-type enzyme. Crystallographic studies did not indicate any gross structural changes in the mutant as compared to the native form. Initial velocity data were best described by a rapid equilibrium ordered mechanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but inhibition by lithium was greatly reduced and became noncompetitive. The pH dependence of catalysis and the solvent isotope effect on kcat are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitrophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. All data taken together are consistent with the hypothesis that modifying or replacing the lysine residue in position 36 decreases its polarizing effect on one of the catalytic metal ions and prevents the efficient deprotonation of the metal-bound water nucleophile.

Base Sequence↗
PubMed1995

Kinetic characterization of enzyme forms involved in metal ion activation and inhibition of myo-inositol monophosphatase.

Activation and inhibition of recombinant bovine myo-inositol monophosphatase by metal ions was studied with two substrates, D,L-inositol 1-phosphate and 4-nitrophenyl phosphate. Mg2+ and Co2+ are essential activators of both reactions. At high concentrations, they inhibit hydrolysis of inositol 1-phosphate, but not 4-nitrophenyl phosphate. Mg2+ is highly selective for inositol 1-phosphate (kcat. = 26 s-1) compared with the aromatic substrate (kcat. = 1 s-1), and follows sigmoid activation kinetics in both cases. Co2+ catalyses the two reactions at similar rates (kcat. = 4 s-1), but shows sigmoid activation only with the natural substrate. Li+ and Ca2+ are uncompetitive inhibitors with respect to inositol 1-phosphate, but non-competitive with respect to 4-nitrophenyl phosphate. Both metal ions are competitive inhibitors with respect to Mg2+ with 4-nitrophenyl phosphate as the substrate. With inositol 1-phosphate, Ca2+ is competitive and Li+ non-competitive with respect to Mg2+. Multiple inhibition studies indicate that Li+ and Pi can bind simultaneously, whereas no such complex was detected with Ca2+ and Pi. Several sugar phosphates were also characterized as substrates of myo-inositol monophosphatase. D-Ribose 5-phosphate is slowly hydrolysed (kcat. = 3 s-1), but inhibition by Li+ is very efficient (Ki = 0.15 mM), non-competitive with respect to the substrate and competitive with respect to Mg2+. Depending on the nature of the substrate, Li+ inhibits by preferential binding to free enzyme (E), the enzyme-substrate (E.S) or the enzyme-phosphate (E.Pi) complex. Ca2+, on the other hand, inhibits by binding to E and E.S, in competition with Mg2+. The results are discussed in terms of a catalytic mechanism involving two metal ions.

Animals↗
PubMed1983

Delicate delegation.

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Personnel Management↗