Search PubMed⌕ Search

Biomedical subjects

R Hensel

Publications and source records attributed to R Hensel.

47 records · Page 3Linked to original sources
PubMed1987

Characterization of two D-glyceraldehyde-3-phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax.

Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP+ and the other for NAD+. NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD+ whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+-specific enzyme) and 49 kDa (NAD+-specific enzyme), respectively. The NADP+-specific enzyme shows significant homology to the known glyceraldehyde-3-phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing. The enzymes are thermostable, the NADP+-specific enzyme with a half-life of 35 min at 100 degrees C, the NAD+-specific enzyme with a half-line of greater than or equal to 20 min at 100 degrees C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60-70 degrees C.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus.

The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75 degrees C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.

Amino Acid Sequence↗
PubMed1983

Affinity labelling of the allosteric site of the L-lactate dehydrogenase of Lactobacillus casei.

Kinetic investigations employing the substrate analogues 2-oxoglutarate and phospho(enol)pyruvate indicate that the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei has a non-catalytic pyruvate-binding site to which, in addition to pyruvate, the allosteric effector fructose 1,6-bisphosphate can also be found. A modification using the 14C-labelled substrate analogue 3-bromopyruvate induces a loss of regulation by fructose 1,6-bisphosphate. The histidine residue labelled by 3-bromopyruvate is homologous to histidine-188 which is part of the anion-binding site of the non-allosteric vertebrate L-lactate dehydrogenases. Thus, the allosteric site of the allosteric L-lactate dehydrogenases corresponds to the anion-binding site of the non-allosteric vertebrate enzymes.

Affinity Labels↗
PubMed1983

The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei.

The polypeptide chain of the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei consists of 325 amino acid residues. Despite the strikingly different enzymatic characteristics of the allosteric L-lactate dehydrogenase of L. casei and of the non-allosteric vertebrate enzymes, the sequence of the allosteric enzyme shows a distinct homology with that of the non-allosteric vertebrate enzymes (average identity: 37%). An especially high sequence homology can be identified within the active center (average identity: 70%). A clear deviation of the L. casei enzyme from the vertebrate enzyme is the lack of the first 12 amino acid residues at the N terminus and an additional 7 amino acid residues at the C terminus. The localization of the binding site of the allosteric effector D-fructose 1,6-bisphosphate and pH and effector-induced changes of the spectroscopic properties are discussed on the basis of the primary structure.

Amino Acid Sequence↗
PubMed1981

Reconstitution of the allosteric L-lactate dehydrogenase from Lactobacillus casei investigated by hybridization.

The reassociation process of the urea-denatured allosteric L-lactate dehydrogenase from Lactobacillus casei was investigated by hybridization experiments between the reassociating enzymes from L. casei and Lactobacillus curvatus. The quantitatively evaluated hybridization patterns indicate an assembly pathway from the unfolded subunits to the tetrameric state via dimers. The comparison of the kinetics of reassociation and reactivation of the L. casei L-lactate dehydrogenase shows that the tetramer is the only active form.

Allosteric Regulation↗
PubMed1981

Amino acid sequence of a dodecapeptide from the substrate-binding region of the L-lactate dehydrogenase from Lactobacillus curvatus, Lactobacillus xylosus and Bacillus stearothermophilus.

The amino acid sequence of dodecapeptides from the substrate-binding region of 3 bacterial L-lactate dehydrogenases (Lactobacillus xylosus, Lactobacillus curvatus and Bacillus stearothermophilus) were determined. They show a very high homology to the sequences of the corresponding known animal enzymes. There is, however, an essential difference between the sequences of pro- and eucaryotic enzymes: the Asn residue in position 166, common to all eucaryotes, is replaced by serine in lactobacilli and by isoleucine in Bacillus stearothermophilus. The cysteine residue in position 165, formerly considered as essential, seems to be restricted to the vertebrates, while all so far investigated invertebrates and bacteria have threonine at this position.

Amino Acid Sequence↗
PubMed1980

Factors affecting the quaternary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as investigated by hybridization and ultracentrifugation.

The allosteric L-lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric from (molecular weight about 145 000) at pH 5.0--5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6-bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35 000) at higher pH values or in the presence of urea. In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatus L-lactate dehydrogenase exists in a dissociation-association equilibrium comprising tetramers, dimers and monomers as indicated by the Sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation. The L-lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH greater than or equal to 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH-dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non-allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.

Allosteric Regulation↗
PubMed1977

Comparative studies of lactate dehydrogenases in lactic acid bacteria. Amino-acid composition of an active-site region and chemical properties of the L-lactate dehydrogenase of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus acidophilus.

The molecular weight, the amino acid composition and the N-terminal and C-terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non-allosteric (L. plantarum, L. acidophilus) L-lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined. The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the the substratebinding site of the L-lactate dehydrogenase dehydrogenase of several animals. However, the 'essential' cysteine residue 165 is replaced by threonine, as it is in the L-lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L-lactate dehydrogenases, but also the allosteric and lactate dehydrogenases from bacterial sources may have evolved from a common gene.

Amino Acids↗
PubMed1977

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. I. Purification and kinetics of the allosteric L-lactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus.

The stability, pH-dependence and kinetic properties of the Mn2+ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiency only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn2+ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.

Allosteric Regulation↗