Enzymic degradation of isotopically labeled compounds

Genovese, Joseph; Schmidt, Klaus; Katz, Joseph

doi:10.1016/0003-2697(70)90097-7

Cited by 15 publications

(4 citation statements)

References 12 publications

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“…1984) and Ara and Rib were chemically degraded using the periodate method (Genovese et al. 1970). [ 14 C]-3-Phosphoglyceric acid derived from DHAP was chemically degraded by the procedure of Andrews et al.…”

Section: Methodsmentioning

confidence: 99%

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

Williams

MacLeod

2006

Photosynth Res

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Abstract14 C-Labelled octulose phosphates were formed during photosynthetic 14 CO 2 fixation and were measured in spinach leaves and chloroplasts. Because mono-and bisphosphates of D-glycero-D-idooctulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified CalvinBenson-Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14 C-labelled D-glycero-D-altro-octulose mono-and bisphosphates were also identified in chloroplasts and leaves.14 CO 2 predominantly labelled positions 5 and 6 of D-glycero-D-ido-octulose 1,8-P 2 consistent with labelling predictions of the modified scheme. The kinetics of 14 CO 2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12 CO 2 rapidly displaced the 14 C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don't rapidly exchange isotopically labelled carbons with unlabelled CO 2 .)A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO 2 assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO 2 fixation at rates in excess of 120 lmol h -1 mg -1 Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and D-glycero-Dido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that 14 C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono-and bisphosphates. However, failure to demonstrate any CO 2 fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido-and altro-octulose. Arabinose 5-phosphate is presented as playing this role...

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Section: Methodsmentioning

confidence: 99%

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

Williams

MacLeod

2006

Photosynth Res

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“…[2-3H]-Glycerol was made by reduction of dihydroxyacetone with NaB3H4 at pH7. sn-[U-14C]Glycerol 3-phosphate was obtained by phosphorylation of [U-14C]glycerol with ATP catalysed by glycerol kinase (EC 2.7.1.30) (Genovese et al, 1970). Enzymes were obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A.…”

Section: Methodsmentioning

confidence: 99%

Pathways of glyceride glycerol synthesis

Rognstad

Clark

Katz

1974

Biochemical Journal

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Isolated rat liver parenchymal cells were incubated for various periods with [U-(14)C,2-(3)H]glycerol and the radioisotopic yields in the major products were determined, as well as the (3)H/(14)C ratios in glyceride glycerol and intracellular glycerol phosphate. Under the conditions used (0.1mm-glycerol+10mm-l-lactate or 10mm-glycerol as substrates), only small differences were found between these (3)H/(14)C ratios. The results suggest a minor role for a pathway of glyceride glycerol synthesis involving reduction of acylated dihydroxyacetone phosphate, under these experimental conditions.

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“…Glycerol was eluted from the borate column with 0.04M-ammonium tetraborate, followed with elution with 0.5 M-acetic acid for glucose. Glycogen glucose was degraded as described by Genovese et al (1970) and Schmidt et al (1970).…”

Section: Analyticalmentioning

confidence: 99%

Lactate metabolism in the perfused rat hindlimb

Shiota¹,

Golden²,

Katz³

1984

Biochemical Journal

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A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.

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Enzymic degradation of isotopically labeled compounds

Cited by 15 publications

References 12 publications

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

Pathways of glyceride glycerol synthesis

Lactate metabolism in the perfused rat hindlimb

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