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Murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate are being explored as a new approach to antagonize the lethal effects of potassium cyanide in mice. Prior studies indicated that these carrier erythrocytes persist in the vascular system for the same length of time as normal erythrocytes and can enhance metabolism of cyanide to thiocyanate. The present studies demonstrate the ability of these carrier red blood cells containing rhodanese and thiosulfate to antagonize the lethal effects of cyanide either alone or in various combinations with sodium nitrite and/or sodium thiosulfate. Potency ratios are compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and carrier erythrocytes containing rhodanese and sodium thiosulfate either alone or in various combinations prior to the administration of potassium cyanide. These results indicate that the administration of carrier erythrocytes containing rhodanese and thiosulfate alone can provide significant protection against the lethal effects of cyanide. These carrier erythrocytes potentiate the antidotal effect of sodium thiosulfate alone or the combination of sodium nitrite and sodium thiosulfate. The mechanisms of cyanide antagonism by these carrier erythrocytes and their broader conceptual significance to the antagonism of other chemical toxicants are discussed.

A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned. into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBLI and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240-and 40-fold, respectively, in the asdglgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase w.ere similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.

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ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data.Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of gigA overlaps with the third base TAA stop codon of the glgC gene.ADPglucose synthetase (EC 2.7.7.27) is an allosteric enzyme in the glycogen biosynthetic pathway of eubacteria (23,24). Among the enteric bacteria, ADPglucose synthetase is activated by glycolytic intermediates with fructose 1,6-bisphosphate as the activator and AMP, ADP, and Pi as inhibitors (23). The enzyme catalyzes the synthesis of ADP glucose from glucose 1-phosphate and ATP in the reaction glucose 1-phosphate + ATP ADPglucose + PPi. This reaction is the first unique step in bacterial glycogen biosynthesis.In Escherichia coli, the structural genes for ADPglucose synthetase (glgC), glycogen synthase (glgA), and branching enzyme (glgB) are mapped at 75 units on the genetic map, flanked by the asd (aspartic semialdehyde dehydrogenase) and glpD (glycerol phosphate dehydrogenase) genes (1). Okita et al. (20) have cloned the structural genes of glycogen biosynthetic enzymes of E. coli K-12 into the PstI site of pBR322, and the nucleotide sequences of the glgC (2), glgB (3), and gigA genes (10) have been determined. Salmonella typhimurium, being closely related to E. coli, shows a lot of similarities in glycogen biosynthesis (12). The ADPglucose synthetases of E. coli and S. typhimurium are similar in that (i) they have similar subunit and native molecular weights; (ii) they have the same spectrum of activators and inhibitors; (iii) they have immunological cross-reactivity; (iv) of the first 27 amino acids in the N terminus 25 are identical; (e) genetically, the glg genes of both are clustered around 75 units on their genetic maps and are cotransducible with asd and glpD genes (28). Recently, we have cloned the glgC and glgA genes from S. typhimurium (14).