Labeling of the active site of aldolase with glyceraldehyde 3-phosphate and erythrose 4-phosphate

Lai, Chun‐Yen; Dretz, G. Martinez-de; Bacila, Metry; Marinello, Enrico; Horecker, B.L.

doi:10.1016/0006-291x(68)90564-0

Cited by 43 publications

(15 citation statements)

References 10 publications

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“…Inhibition of the enzyme resulting from prolonged incubation with excess of substrate has been observed by others (Woodfin, 1967;Lai, Martinez-de Dretz, Bacila, Marinello & Horecker, 1968). We find that this inhibition is accompanied by decreased thiol group reactivity towards DTNB (Table 3).…”

Section: Naturesupporting

confidence: 86%

The reactivity of thiol groups and the subunit structure of aldolase

Anderson

Perham

1970

Biochemical Journal

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1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.

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Section: Naturesupporting

confidence: 86%

The reactivity of thiol groups and the subunit structure of aldolase

Anderson

Perham

1970

Biochemical Journal

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“…Only high concentrations (6 mM) of this compound were inhibitory; the isoenzymes were not affected by 0.6 MM D-erythrose-4-P. The pea leaf aldolases are less sensitive to inhibition by L-glyceraldehyde-3-P and erythrose-4-P than the rabbit muscle aldolase (11) but more sensitive than the kidney and liver enzymes (1,11).…”

Section: Resultsmentioning

confidence: 94%

“…D-Glyceraldehyde-3-P inhibits the enzymes, but, since our aldolase preparations were contaminated with triose-P isomerase, it was not possible to study inactivation quantitatively with this stereoisomer. The rabbit muscle enzyme is inhibited by both isomers (11). Inhibition by D-erythrose-4-P was mixed (and varied between particulate chloroplast (500,OOOg precipitate, 0, dotted line) and cytoplasmic (A, solid line) aldolase catalyzed cleavage of fructose-1,6-diP.…”

Section: Resultsmentioning

confidence: 99%

Chloroplast and Cytoplasmic Enzymes

Anderson

Pacold

1972

Plant Physiol.

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Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. In mammalian systems fructose-1,6-diP aldolases from different tissues have kinetic properties related to the function (synthetic or degradative) of the enzyme. Differences in the ratio V.a. fructose-1,6-diP cleavage to Vma. fructose-1,6-diP synthesis led Rutter and coworkers (15) to conclude that muscle aldolase is "more effective in cleaving fructose-1 ,6-diP whereas liver aldolase is relatively more effective in synthesis." Since in green plants chloroplast and cytoplasmic aldolases might also be expected to have opposing functions, it seemed of interest to compare the properties of the plant aldolases. Although spinach whole leaf (8, 14) and chloroplast (7) aldolases and a pea seed aldolase (10,19,20) had been studied prior to the present investigation, no higher plant aldolase known to be cytoplasmic had been characterized. The purpose of the experiments reported here was to characterize the pea leaf chloroplast and cytoplasmic aldolases and to compare the physical and kinetic properties of the isoenzymes.The results indicate that the chloroplast and cytoplasmic aldolases are exceedingly similar enzymes, the pH optima and Michaelis constants for fructose-1 ,6-diP, fructose-1-P, and sedoheptulose-1,7-diP being almost identical. Isoelectric Focusing. Chloroplast and cytoplasmic extracts, prepared as described previously (5) (except that the chloroplast extract was centrifuged 45 min at 500,000g), and containing 10 to 15 mg of protein, were subjected to electrophoresis for 1.5 days in pH 3 to 6 ampholytes at 450 v or pH 4 to 6 ampholytes at 500 v, in a 100-ml LKB isoelectric focusing column at 10 C. Ampholyte and electrode solutions were made up with 5 mm 2-mercaptoethanol. The anode solution (made with 0.5 N H2SO4) was at the bottom of the column. The cathode solution was 0.25 N NaOH. The density gradient was usually made with sucrose. Sorbitol was used in place of sucrose in experiments where aldolase activity was measured as fructose-1,6-diP formation. Fractions (9 drops, 0.7 ml) were collected and analyzed for enzyme activity. The pH of the individual fractions was measured at 0 C using a Radiometer pH meter 26.Purification of Chloroplast and Cytoplasmic Aldolases. Chloroplast and cytoplasmic extracts were prepared as described previously (5) except that the procedure was scaled up for 1 kg of tissue, and the entire shoot of the plant was used. Chloroplasts were usually prepared beforehand and stored at -20 C. The method of Fluri et al. (8) for purification of spinach aldolase was followed, with some modifications. The chloroplast extract, in 10 mm potassium phosphate buffer, pH 7.5, was brought to pH 5.5 with 3.5 M acetic acid, and the supernatant fractio...

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“…4A). These two compounds have been reported to reduce the activity of aldolase (22) and aspartate aminotransferase (25), and in the latter case Nisselbaum et al (25) pointed out that the compounds possess both free aldehyde and phosphoryl groups which may have been required for the observed effect; higher homologues (ribose-5-P and glucose-6-P) which exist predominantly in the internal hemiacetal configuration had no effect. In the present experiments, the pea shoot nonreversible D-G3P dehydrogenase was only slightly inhibited by D-ribose-5-P (Fig.…”

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confidence: 98%

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues

Kelly

Gibbs²

1973

Plant Physiol.

114

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Preparations of TPN-linked nonreversible D-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9), free of TPN-linked reversible D-glyceraldehyde 3-phosphate dehydrogenase, have been obtained from green shoots, etiolated shoots, and cotyledons of pea (Pisum sativum), cotyledons of peanut (Arachis hypogea), and leaves of maize (Zea mays). The Plant tissues contain three enzymes for the oxidation of D-G3P' (12). Two of these catalyze a Pi-dependent, reversible oxidation of D-G3P to 1,3-diP-glycerate; one enzyme is DPNspecific and found in both photosynthetic and nonphotosynthetic tissues, while the other uses TPN and is most probably confined to the chloroplast. Both enzymes have been extensively studied (29). The third enzyme, which is TPN-specific, oxidizes D-G3P to glycerate-3-P in a reaction which does not require Pi and is not reversible. Evidence for this enzyme activity in green leaves was reported by leaves. Recently, Jacob and d'Auzac (17) have purified the enzyme from the latex of Hevea brasiliensis. In this laboratory, the partially purified enzyme from pea shoots was inhibited strongly by L-G3P (19). It is possible that the scarcity of reports on the enzyme is related to the presence of this inhibitor in commercial preparations of the substrate D-G3P.In the present investigation, the significance of this nonreversible D-G3P dehydrogenase to plant carbohydrate metabolism has been evaluated. A preparation of pea shoot enzyme, free of reversible Pi-dependent D-G3P dehydrogenase activity, has now been obtained, and the properties have been compared with those of the same enzyme partially purified from several other plant tissues. In addition, a relatively specific assay for the determination of the enzyme activity in cell-free extracts is described. Changes in the enzyme activity during the germination of peas and castor beans and the distribution of the enzyme in representative tissues were examined.The properties and in vivo localization of the three D-G3P dehydrogenases were compared, and the results indicate that, in some tissues, a major portion of triose-P oxidation may be mediated by the nonreversible enzyme.MATERIALS AND METHODS Materials. Pea (Pisum sativum L. var. Progress No. 9), maize (Zea mays L. var. Early Fortune), peanuts (Arachis hypogea L. var. Jumbo Virginia), and castor bean (Ricinus communis L.) were grown under natural lighting in a glasshouse at 16 to 18 C or in the dark at 22 C.The following chemicals and enzymes were obtained from the Sigma Chemical Company: ATP, dihydroxyacetone-P, DPN, D-erythrose-4-P, D-fructose-1 ,6-diP, DL-G3P, D-glycerate-3-P, P-glycolate, P-hydroxypyruvate, D-ribose-5-P, TPN, TPNH, G3P dehydrogenase, 3-P-glycerate kinase, and triose-P isomerase. Chloroacetol-P was a gift of Dr. F. C. Hartman through the courtesy of Dr. Louise Anderson. All chemicals were prepared as sodium salts at the recommended pH before use.L-G3P was prepared according to the method of Venkataraman and Racker (38). The D-G3P present in 0.65 mmole of DL-G3P was converted to glyce...

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Labeling of the active site of aldolase with glyceraldehyde 3-phosphate and erythrose 4-phosphate

Cited by 43 publications

References 10 publications

The reactivity of thiol groups and the subunit structure of aldolase

The reactivity of thiol groups and the subunit structure of aldolase

Chloroplast and Cytoplasmic Enzymes

Nonreversible d-Glyceraldehyde 3-Phosphate Dehydrogenase of Plant Tissues

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