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Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

ABSTRACT: 1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.

SUBMITTER: Baldwin JE

PROVIDER: S-EPMC1161726 | biostudies-other | 1980 Mar

REPOSITORIES: biostudies-other

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Project description:1. [2-(3)H,U-(14)C]- or [3-(3)H,U-(14)C]-Lactate was administered by infusion or bolus injection to overnight-starved rats. Tracer lactate was injected or infused through indwelling cannulas into the aorta and blood was sampled from the vena cava (A-VC mode), or it was administered into the vena cava and sampled from the aorta (V-A mode). Sampling was continued after infusion was terminated to obtain the wash-out curves for the tracer. The activities of lactate, glucose, amino acids and water were followed. 2. The kinetics of labelled lactate in the two modes differed markedly, but the kinetics of labelled glucose were much the same irrespective of mode. 3. The kinetics of (3)H-labelled lactate differed markedly from those for [U-(14)C]lactate. Isotopic steady state was attained in less than 1h of infusion of [(3)H]lactate but required over 6h for [U-(14)C]lactate. 4. (3)H from [2-(3)H]lactate labels glucose more extensive than does that from [3-(3)H]lactate. [3-(3)H]Lactate also labels plasma amino acids. The distribution of (3)H in glucose was determined. 5. Maximal radioactivity in (3)HOH in plasma is attained in less than 1min after injection. Near-maximal radioactivity in [(14)C]glucose and [(3)H]glucose is attained within 2-3min after injection. 6. The apparent replacement rates for lactate were calculated from the areas under the specific-radioactivity curves or plateau specific radioactivities after primed infusion. Results calculated from bolus injection and infusion agreed closely. The apparent replacement rate for [(3)H]lactate from the A-VC mode averaged about 16mg/min per kg body wt. and that in the V-A mode about 8.5mg/min per kg body wt. The apparent rates for [(14)C]lactate (;rate of irreversible disposal') were 8mg/min per kg body wt. for the A-VC mode and 5.5mg/min per kg body wt. for the V-A mode. Apparent recycling of lactate carbon was 55-60% according to the A-VC mode and 35% according to the V-A mode. 7. The specific radioactivities of [U-(14)C]glucose at isotopic steady state were 55% and 45% that of [U-(14)C]lactate in the A-VC and V-A modes respectively. We calculated, correcting for the dilution of (14)C in gluconeogenesis via oxaloacetate, that over 70% of newly synthesized glucose was derived from circulating lactate. 8. Recycling of (3)H between lactate and glucose was evaluated. It has no significant effect on the calculation of the replacement rate, but affects considerably the areas under the wash-out curves for both [2-(3)H]- and [3-(3)H]-lactate, and calculation of mean transit time and total lactate mass in the body. Corrected for recycling, in the A-VC mode the mean transit time is about 3min, the lactate mass about 50mg/kg body wt. and the lactate space about 65% of body space. The V-A mode yields a mass and lactate space about half those with the A-VC mode. 9. The area under the wash-out curve for [(14)C]lactate is some 20-30 times that for [(3)H]lactate, and apparent carbon mass is 400-500mg/kg body wt. and presumably includes the carbon of glucose, pyruvate and amino acids, which are exchanging rapidly with that of lactate.

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Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

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