In a previous study by the authors, two isoenzymes of 6-phosphogluconate dehydrogenase were isolated from cultures of tobacco tissue Nicotiana tabacum W-38 and shown to be similar in their pH optima and MWs and in their affinities toward 6-phosphogluconate or NADP+. In an attempt to clarify the structural relationships between these two isoenzymes, peptide mapping of trypsin digests of the purified isoenzymes was performed. The maps were found to be similar, with at least 29 peptide groups from the trypsin digestion of each isoenzyme being alike. There were, however, definite minor differences in the peptide maps of the two isoenzymes.

Anodic polyacrylamide gel electrophoresis of extracts of cultures of tobacco tissue Nicotiana tabacum W-38 revealed the presence of two 6-phosphogluconate dehydrogenases (6PGD). The slow and the fast anodic migrating zones were designated I and II, respectively. After purification, enzymes from both zones exhibited no major differences in their affinity towards 6-phosphogluconate (6PG) or NADP+, and were found to have approximately the same pH optima and MWs (69 000–72 000). The coumarins scopoletin and esculetin showed some inhibitory effect on each isozyme at 0.4 mM. Below 0.3 mM, however, esculetin stimulated the activity of zone I when lower amounts of 6PG (S0.25) were used. The glucosylated compounds, scopolin and esculin, were much more inhibitory towards the 6PGDs than their respective aglycones. Ferulic, p-coumaric and caffeic acids seemed to have an inhibitory effect dependent on 6PG concentration. A larger inhibition was observed in each case at the lower 6PG levels used. Zone I activity appeared to be inhibited to a greater degree than zone II activity by 0.4 mM p-coumaric acid with low 6PG. Of the phenolic compounds tested, chlorogenic acid was most effective, completely inhibiting the enzyme activity at 0.4 mM. Of the non-phenolic compounds investigated, glucose 1,6-diphosphate inhibited both isoenzymes of 6PGD at lower 6PG concentrations. On the other hand, 2,3-diphosphoglycerate activated both isoenzymes up to 200% of their original activity.

Two isoperoxidases (A f and C n ) from the medium of tobacco tissue suspension culture WR-132 grown in darkness have been purified to apparent homogeneity and partially characterized. C n and A f have MWs of ca 30 000 and 54 000, respectively. A f has ca 5.1% carbohydrate, but none could be detected in C n . Both isoperoxidases appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The K m s for guaiacol are 4 and 13.3 mM for A f and C n , respectively, while both isoperoxidases have a pH optimum at 6.5. C n , is dissimilar to other isoperoxidases from tobacco tissue cultures, but A f is very similar to isoperoxidase A 3 from W-38 tobacco tissue culture.

Eight isoperoxidases from tobacco suspension culture WR-132 (termed Cn, C3, C4, Ac, and Af) and tobacco callus culture W-38 (termed A1, A2, and A3) have been subjected to trypsin digestion followed by peptide mapping. The peptide maps of isoperoxidases Af and A3 are identical. All other isoperoxidases do not appear to be dramatically dissimilar in certain portions of their sequence, since many matching peptides have been found when various isoperoxidases are cross-compared. However, only two, and possibly three, highly homologous peptides are present in all of the isoperoxidases.

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP + , with K m 's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K m of 0.31 mM. The NADP + double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K m and V max values with increasing NADP + , denoting negative cooperativity. The two K m 's for high and low NADP + concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP + binding sites.

In order to study the effect of light on the tobacco tissue culture WR-132, 5 passages (10 days' growth per passage) of these cells were grown in darkness, and 3 passages were separately grown in intense light (16000 lx). All other growth conditions were the same. The resulting isoperoxidase patterns present in these cells and in their growth media were analyzed at 2-day intervals during this period and then compared with the isoperoxidase patterns of cells grown under dim light conditions (10 lx). A new cathodic isoperoxidase (Cn) appeared in the medium within 2 days after the cells were placed in the dark. Cn was present in all media of WR-132 cell cultures analyzed throughout the 5 passages grown in darkness. The fifth passage in darkness produced total cessation of growth (apparent death). Cn increased and new anodic isoperoxidases Aa, Ab, Ad and Ae appeared in the media as the cells approached death in darkness.

AbstractIsoperoxidase A3 from tobacco tissue culture, Nicotiana tabacam W‐38, apparently does not have a subunit structure. Its molecular weight is approximately 54,000.The oxidation of scopoletin appears to occur in more than a single step since a blue intermediate is first formed. This is followed by the production of the final yellow product. A plausible explanation for this observation is the coupling of free radicals generated by the primary enzyme catalyzed reaction.

Two anodic isoperoxidases (A 1 and A 2 ) from tobacco tissue culture W-38 and two cathodic isoperoxidases (C 3 and C 4 ) from tobacco suspension culture WR-132 have been separated and characterized. Molecular weights for each of the isoperoxidases have been determined by two different methods. Only C 4 contained a carbohydrate component. The substrate specificity and the pH optima for the four enzymes with each of five substrates were determined.

Scopoletin was found to be a substrate for a single anodic isoperoxidase isolated from tobacco callus tissue W-38. Isolation of this peroxidase was accomplished using DEAE-cellulose chromatography. This isoperoxidase catalysed the destruction of scopoletin in the presence of H 2 O 2 only. An enzyme assay for the scopoletin reaction was developed. The pH optimum of the enzyme was 5·5 and the apparent K m s for scopoletin and H 2 O 2 were 0·6 and 0·9 rnM respectively.

An anodic isoperoxidase (A2) from tobacco tissue culture W-38 and a cathodic isoperoxidase (C4) from tobacco tissue suspension culture WR-132 have been separated and characterized. Both isoperoxidases catalysed oxidation of ferulic acid in the presence of H2O2. When the reaction mixture was subjected to TLC, ferulic acid was found to have been converted to an unknown compound which, after treatment with ammonia, fluoresces green in UV light. Both the isoperoxidases A2 and C4 appear to follow simple Michaelis-Menten kinetics with respect to guaiacol as the substrate. The Kms for guaiacol are 4 and 4·5 mM for isoperoxidases C4 and A2, respectively. The pH optimum for both enzymes is about 6·0. The effect of various phenolic and related compounds on the activity of each isoperoxidase is reported and discussed.

1.1. The kinetics and regulation by effectors of glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) from WI-38 and SV40-transformed WI-38 (WI-38VA13A) were studied.2.2. Both the apparent Km for NAD+ (about four times greater) and the interaction between NAD+ binding sites appeared greater for the enzyme of the SV-40 transformed cells. A consequence of this was a fully active WI-38 enzyme in vitro, when the enzyme from WI-38VA13A would be approximately 50 per cent active.3.3. The WI-38VA13A enzyme was more susceptible to inhibition by adenine nucleotidcs.

1. Calciferous spicules were obtained fromBriareum asbestinum, a gorgonian or “soft” coral.2. The spicules were washed extensively and treated with potassium hydroxide to remove the exterior sheath and any proteins on the outside.3. After solubilization of the calcium carbonate with acid, an insoluble material remained.4. Peptides with molecular weights between 1600 and 5000 were isolated from this core material.5. These peptides had amino acid compositions characteristic of the collage like proteins.

The effect of indoleacetic acid, 2,4-dichlorophenoxyacetic acid, tryptophan, scopoletin, scopolin, and related compounds on the activity of a phenylalanine ammonia-lyase preparation from tobacco tissue WR-132 grown in suspension culture has been investigated. Under the experimental conditions used, IAA and its probable plant precursor trytophan, were found to inhibit the enzyme strongly. Scopoletin showed slightly lesser inhibition. The synthetic auxin, 2,4-D, produced little effect on the enzyme except at the higher concentration used. Scopolin, kinetin, gibberellic acid, and β-indole-3-propionic acid showed no effect. The concentration of β-mercaptoethanol used in each assay was found to affect the values obtained for the percentage inhibition of phenylalanine ammonia-lyase by IAA. The possible physiological significance of these inhibitions is discussed.

Time studies were made on the fate of scopoletin-4-14C taken up through the roots of whole tobacco seedlings. Of the total radioactivity recovered in one set of scopoletin-4-14C continuous feeding experiments, a range of 43–51% (av. 46%) was found to be in the 'soluble' portion of the roots and shoots. At one period (after 4 hr of continual feeding of scopoletin-4-14C), about 91% of the radioactivity in the 'soluble' fraction was found to be present as scopoletin-7-glucoside (scopolin); less than 3% as scopoletin; and approximately 5% as fabiatrin. The percentage of the total recovered radioactivity contributed by the 'soluble' compounds decreased with time, whereas the percentage of the radioactivity due to the 'insolubles' increased with time. The results also suggest that some of the radioactive scopolin (from scopoletin-4-14C not converted at an early stage to 'insoluble' material) may be converted with more time to other 'soluble' compounds and possibly also to some 'insoluble' material. In the roots, fabiatrin was formed slowly from scopolin. The half-life of scopolin was found to be 16 days. Fabiatrin had a slower turnover rate than did scopolin. No measurable amount of fabiatrin was found in the shoots. When a lower starting concentration of scopoletin-4-14C was fed to the seedling in continuous feeding experiments, a larger percentage of the radioactivity appeared in the 'insoluble' fraction of tobacco roots. A hypothesis based on compartmentation of metabolic and vacuolar pools is proposed.

Pyruvate kinase (E. C. 2.7.1.40) from human embryonic lung tissue, WI-38, has a pH optimum range of 7.0 to 7.2. This enzyme was found to be activated 2.1, 5.0 and 2.5 fold by fructose-1,6-diphosphate, KC1, and NH4Cl, respectively, and unaffected by NaCl. Even though the degree of activation by these effectors is different, they all cause an increase in maximal velocity and increase the affinity of the enzyme for phosphoenolpyruvate. ATP and L-alanine were found to inhibit maximal velocity by 40% and 70%, respectively, and in a non-competitive manner with respect to both substrates. Speculation is offered on the physiological effect of these modulators on glycolysis and glyconeogenesis.

Journal Article Glutamine Synthetase from Nocardia corallina Get access Shingo KAJINAMI, Shingo KAJINAMI Department of Chemistry, University of OklahomaNorman, Oklahoma 73069 Search for other works by this author on: Oxford Academic PubMed Google Scholar Eddie C. SMITH Eddie C. SMITH Department of Chemistry, University of OklahomaNorman, Oklahoma 73069 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 69, Issue 2, January 1971, Pages 429–432, https://doi.org/10.1093/oxfordjournals.jbchem.a129483 Published: 01 January 1971 Article history Received: 01 July 1970 Published: 01 January 1971

The pH optimum for glucose-6-phosphate dehydrogenase from tobacco tissue culture was determined to be between 8 and 9. The plot of the reaction velocity vs. glucose-6-phosphate concentration showed a non-sigmoidal, hyperbolic saturation curve. A similar curve was obtained for the NADP + saturation function. Compounds that may be related to glucose-6-phosphate metabolism were tested for their effects on the enzyme. It was found that the phenolic compounds, scopoletin, scopolin, esculin and ferulic acid, inhibited the enzyme. Similar inhibitions by scopolin and scopoletin were observed for three other NADPH producing dehydrogenases from the same plant cells; namely, 6-phosphogluconate dehydrogenase, NADP + specific isocitrate dehydrogenase and NADP + specific malate dehydrogenase.

Under the described experimental conditions, lactate dehydrogenase (LDH) of Helisoma antrosum is characterized as follows: 1.1. At low substrate (lithium lactate) concentrations (0·01–0·03 M), LDH of whole snail homogenates appears to display hyperbolic kinetics. At higher substrate concentrations (0·04–0·16 M), LDH displays sigmoid kinetics.2.2. LDH exists as a multiple molecular form displaying five isoenzymes.3.3. A Hill plot of the data for whole snail homogenates indicates that at least one of the LDH isoenzymes possesses at least two interacting binding sites.4.4. Isoelectric focusing demonstrated four molecular forms of LDH, the isoelectric points (pI) of these forms being 3·8, 5·2, 6·2 and 8·5.5.5. The Michaelis constant (apparent Km) of the LDH from whole snail homogenates is 5·5 × 10−2 M.6.6. LDH-4, and possibly LDH-5, correspond to a thick band which had been previously demonstrated with amino black (protein stain) and Ponceau red (lipoprotein stain).7.7. LDH-4 is the predominant band in acrylamide gels.