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Biomedical subjects

H Grisebach

Publications and source records attributed to H Grisebach.

At least 37 records · Page 2Linked to original sources
PubMed1982

Enzymic synthesis of lignin precursors. Purification and properties of 4-coumarate:CoA ligase from cambial sap of spruce (Picea abies L.).

4-Coumarate:CoA ligase was purified from cambial sap of spruce (Picea abies). A 1627-fold purification of the enzyme with a yield of 37% was achieved by a six-step procedure including dye-ligand chromatography. Isozymes of the ligase were not detected. The enzyme has an Mr of about 63 000 and is a single polypeptide chain. Ferulic, 4-coumaric and caffeic acids are efficient substrates for the ligase. In contrast to some ligases from angiosperms, the ligase from spruce (gymnosperm) does not activate sinapic acid. The substrate specificity of the ligase is consistent with the lignin composition of spruce.

Coenzyme A Ligases↗
PubMed1981

Enzymic synthesis of lignin precursors. Comparison of cinnamoyl-CoA reductase and cinnamyl alcohol:NADP+ dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.).

Cambial sap of spruce (Picea abies) proved to be a good source for isolation of cinnamoyl-CoA reductase and cinnamyl alcohol:NADP+ dehydrogenase. Apparently homogeneous enzymes were obtained by a multistep procedure including dye-ligand chromatography and for the reductase also affinity chromatography on (coenzyme A)-agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported. Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean. Reduction of feruloyl-CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 x 10(-4) M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the Km-value of feruloyl-CoA in phosphate buffer (5.2 microM) is about 14-times similar than in citrate buffer (73 microM). Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources whereas sinapoyl-CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase. 4-Coumaroyl-CoA is a poor substrate for the reductase from both spruce and soybean. This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.

Alcohol Oxidoreductases↗
PubMed1980

Biosynthesis of streptomycin. Purification and properties of a dTDP-L-dihydrostreptose: streptidine-6-phosphate dihydrostreptosyltransferase from Streptomyces griseus.

dTDP-L-dihydrostreptose: streptidine-6-phosphate dihydrostreptosyltransferase, an enzyme involved in the biosynthesis of streptomycin, has been purified from Streptomyces griseus to near homogeneity by a six-step procedure involving chromatography on streptidine-6-phosphate-Sepharose. By gel filtration the apparent Mr of the enzyme was found to be about 63 000. The subunit Mr found on sodium dodecylsulfate gels is about 35 000. The transferase is dependent on Mn2+ or Mg2+ ions. Co2+ is as effective as Mg2+. From the substrates tested only streptidine 6-phosphate was an acceptor for dihydrostreptose in the synthesis of O-alpha-L-dihydrostreptose(1 leads to 4)-streptidine 6-phosphate. No activity was found with streptidine, 2-deoxystreptamine and 4-deoxy-streptamine. The activity of the transferase in the course of fermentation runs parallel to the activity of dTDP-dihydrostreptose synthase and reaches a maximum after around 50 h of fermentation, just before appearance of streptomycin in the medium.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Biosynthesis of streptomycin. Enzymatic formation of dihydrostreptomycin 6-phosphate from dihydrostreptosyl streptidine 6-phosphate.

Incubation of O-alpha-L-dihydrostreptose (1 leads to 4) streptidine 6-phosphate (I) with a protein-free extract from Streptomyces griseus as endogenous donor and a cell-free extract from this organism led to formation of dihydrostreptomycin 6-phosphate (II). The product was identified by paper chromatography and by its degradation to dihydrostreptobiosamine (III). II was not formed when either the donor solution or the dialysed cell-free extract was omitted. The results corroborate the role of I as intermediate in streptomycin biosynthesis. The synthesis of I from dTDP-L-dihydrostreptose, streptidine 6-phosphate and a dihydrostreptosyltransferase from S. griseus has been shown previously.

Chromatography, Paper↗
PubMed1979

Biosynthesis of streptomycin. Enzymic oxidation of dihydrostreptomycin (6-phosphate) to streptomycin (6-phosphate) with a particulate fraction of Streptomyces griseus.

Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100,000 X g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreducatase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin.

Centrifugation, Density Gradient↗
PubMed1979

Biosynthesis of streptomycin. dTDP-dihydrostreptose synthase from Streptomyces griseus and dTDP-4-keto-L-rhamnose 3,5-epimerase from S. griseus and Escherichia coli Y10.

dTDP-dihydrostreptose synthase from Streptomyces griseus was purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-L-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-D-glucose was determined to be 25 microM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67,000 and 32,000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-L-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy-[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-6-rhamnose as a free intermediate in the epimerase reaction are reported.

Carbohydrate Epimerases↗
PubMed1979

Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures.

Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.

Alcohol Oxidoreductases↗
PubMed1977

UDP-apiose/UDP-xylose synthase. Subunit composition and binding studies.

The UDP-apiose/UDP-xylose synthase from cell suspension cultures of parsley has been purified 1400-fold by an improved method. The ratio of apiose to xylose formed from UDP-D-glucuronic acid (UDP-GlcUA) remained constant throughout the purification procedure. Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE-cellulose at different pH-values, and on omega-aminoalkyl-Sepharose, and by gel filtration on Acrylex P-100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N-terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids. Separation of the two proteins was achieved on DEAE-cellulose in the presence of urea. After dialysis only the 86000-Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000-Mr protein enzyme stability was decreased drastically. By equilibrium dialysis it was found that 0.5 mol UDP-GlcUA are bound per mole of 86000-Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4-keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C-3 of the 4-ulose intermediate prior to ring contraction to apiose was found.

Carboxy-Lyases↗
PubMed1976

Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax).

A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.

Aldehyde Oxidoreductases↗