Enzymic degradation of isotopically labeled compounds

Schmidt, Klaus; Genovese, Joseph; Katz, Joseph

doi:10.1016/0003-2697(70)90098-9

Cited by 30 publications

(6 citation statements)

References 6 publications

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“…Hexoses were enzymatically degraded using a method based on that of Schmidt et al (1970). Between 1-10 p.mol of glucose can be completely converted to 3-phosphoglycerate (3PGA) and glycerol-3-phosphate by incubating in (end volume 2 ml) 300 mM TEA (pH 7.4) containing 30 mM MgC12, a threefold excess of ATP over glucose, NAD § equivalent to 1% of the glucose added, 20 mM NA-arsenate, 2 mM dithiothreitol, 250 ~tM 2-phosphoglycolate, 2.5 U hexokinase, 10 U phosphoglucoisomerase, 5 U phosphofructokinase, I U aldolase, 5 U glycerol-3-phosphate dehydrogenase, and 8 U glyceraldehyde-3-phosphate dehydrogenase for 2-3 h at 25 ~ C. The mixture was then heated to 95 ~ C for 10 min, cooled and centrifuged.…”

Section: Methodsmentioning

confidence: 99%

A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers, and maize endosperm

Hatzfeld

Stitt

1990

Planta

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We have investigated whether starch accumulation in heterotrophic cell-suspension cultures of Chenopodium rubrum L., developing potato (Solarium tuberosum L.) tubers or maize (Zea mays L.) endosperm involves import of triose phosphates or of hexose units into the plastid, and whether there is a rapid recycling of triose phosphates back to hexose phosphates in the cytosol of these tissues, (i) Cell suspensions, potato tubers or intact maize kernels were supplied with [1-(14)C] glucose or [6-(14)C]glucose. The glucosyl residues from starch were isolated and degraded by an enzymic procedure to determine how much radioactivity had been redistributed into the opposite half of the glucose molecule. Randomisation was incomplete, affecting only 18%-38% of the molecules in Chenopodium, 16%-26% of the molecules in potato, or 30% of the molecules in maize. It is concluded that the major route for starch synthesis involves import of hexose units, (ii) The glucosyl and fructosyl moieties of sucrose were isolated and degraded to determine the extent of recycling in the cytosol. There was significant randomisation, lying between 30%-40% in Chenopodium, 20%-26% in potato, and 8%-12% in maize. It is concluded that there is considerable recycling of triose phosphates in the cytosol. (iii). Sucrose from cells supplied with [1-(14)C]glucose was more randomised than sucrose from cells supplied with [6-(14)C]glucose. This is explained in terms of the oxidative pentose-phosphate pathway (iv). Equations are used to estimate the rate of recycling from triose phosphates to hexose phosphates in the cytosol. The estimated rate of recycling is considerably larger than the net glycolytic flux or the activity of the oxidative pentose-phosphate cycle.

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Section: Methodsmentioning

confidence: 99%

A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers, and maize endosperm

Hatzfeld

Stitt

1990

Planta

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“…Glucose degradation was carried out after dismutation to 3-phosphoglycerate and sn-glycerol 3-phosphate (Schmidt et al, 1970). The two compounds were separated by ion-exchange chromatography and the specific radioactivity of each was determined (Katz et al, 1976a).…”

Section: Methodsmentioning

confidence: 99%

Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats. The use of 14C-labelled lactate in studies of hepatic gluconeogenesis

Grunnet

Katz

1978

Biochemical Journal

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1. Hepatocytes from starved rats were incubated with l-lactate and NH(4)Cl or norvaline, and the rates of the tricarboxylic acid cycle and of gluconeogenesis were calculated from changes in metabolite concentrations or from radioisotopic data from incubations with labelled lactate or propionate. 2. Gluconeogenesis was stimulated by the addition of 10mm-NH(4)Cl, 5mm-norvaline or 1mm-oleate by 27, 45 and 59% respectively. NH(4)Cl or norvaline also increased lactate uptake. Norvaline inhibited urea synthesis from NH(4)Cl by 85%. 3. The effects of NH(4)Cl and norvaline were not additive. However, NH(4)Cl inhibited and norvaline was without effect on gluconeogenesis from pyruvate, indicating that the two compounds act by different mechanisms. 4. The tricarboxylic acid-cycle flux was increased 80% by lactate, and NH(4)Cl caused a further 25% stimulation. Norvaline had no effect on the tricarboxylic acid-cycle flux. NH(4)Cl and norvaline tripled and doubled, respectively, flux through pyruvate dehydrogenase. 5. Total ATP formation was calculated to range from 470 to 830mumol/h per 100mg of protein, of which the basic metabolic activity accounted for 400-450mumol/h per 100mg of protein. ATP formation does not seem to be rate-limiting for gluconeogenesis. 6. Pyruvate recycling was estimated from the (14)C yield from [1-(14)C]propionate in lactate and glucose to be 10-30% of the flux of phosphoenolpyruvate to glucose. The further addition of NH(4)Cl more than doubled the recycling of pyruvate. 7. [1,4-(14)C]Succinate was rapidly metabolized by hepatocytes. About 20% of the radioactivity was recovered in glucose, indicating that succinate is also metabolized by intact (non-damaged) hepatocytes. 8. It is concluded that the metabolism of lactate by the liver is too complex to allow simple rate measurements with labelled compounds.

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“…The coupled reactions had the advantage of recycling NAD ϩ , and thus the coenzyme concentration could be kept constant. In addition, the irreversibility of the reaction was attained by incubation in 20 mM arsenate (35).…”

Section: Assay Conditions For the Measurement Of The Isotope Effects mentioning

confidence: 99%

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

Gleixner

Schmidt

1997

Journal of Biological Chemistry

120

107

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The kinetic and equilibrium isotope effects on the fructose-1,6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13 C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13 C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13 (V max /K m ) on C-3 was 1.016 ؎ 0.007 and 0.997 ؎ 0.009 on position C-4, and the equilibrium isotope effects K 12 /K 13 on these positions were 1.0036 ؎ 0.0002 and 1.0049 ؎ 0.0001.The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13 C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13 C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13 C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.The relative enrichment of carbon-13 in the carboxyl group of amino acids, as observed by Abelson and Hoering (1) in 1961, was the first indication for the existence of non-statistical isotope distributions in biological compounds. Later results on acetic acid (2) and on acetoin (3) gave evidence that this observation was just one example of a common phenomenon. In order to find a general explanation for this observation Galimov (4) discussed that even in chemically unequilibrated systems, e.g. biological systems, a microscopic reversibility in enzymatic reactions is the origin for a thermodynamically ordered isotope distribution. The author's calculations could in fact explain some of the isotopic patterns of natural compounds known at that time. However, the presumption of a general thermodynamic equilibrium in biological systems is probably not realistic. In our opinion kinetic isotope effects on enzymatic reactions should be considered as primary causes for isotope discriminations. This has been proven for the primary CO 2 -fixing reactions (5-8). In secondary metabolism, the isotope effect on the pyruvate dehydrogenase reaction has been made responsible for the general depletion of 13 C in metabolites of acetyl-CoA, such as fatty acids or isoprenoids (9 -15).More detailed interpretations were possible when the total isotopic patterns of primary and secondary metabolites became available. Especially, our corresponding investigations on glucose indicated an enrichment of 13 C in positions C-3 and C-4, and a depletion of 13 C in positions C-1 and C-6 (Fig. 1) of this important primary metabolite (16). We have a...

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Enzymic degradation of isotopically labeled compounds

Cited by 30 publications

References 6 publications

A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers, and maize endosperm

A study of the rate of recycling of triose phosphates in heterotrophic Chenopodium rubrum cells, potato tubers, and maize endosperm

Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats. The use of 14C-labelled lactate in studies of hepatic gluconeogenesis

Carbon Isotope Effects on the Fructose-1,6-bisphosphate Aldolase Reaction, Origin for Non-statistical 13C Distributions in Carbohydrates

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