The effect of zinc ions on carbohydrate metabolism and intracellular Zn2+ was studied in hepatocytes from fed rats. The addition of ZnCl2 to the medium led to an almost 3-fold increase in lactate production and an increase in net glucose production of about 50%. Half-maximal rates occurred at about 40 microM ZnCl2. These effects were not seen with Mn2+, Co2+, or Ni2+ up to 80 microM, whereas Cu2+ at 80 microM and Cd2+ or Pb2+ at 8 microM exhibited similar effects as 80 microM ZnCl2. Changes in intracellular Zn2+ were followed by single cell epifluorescence using zinquin as a specific probe. Intracellular free Zn2+ in isolated hepatocytes was 1.26 +/- 0.27 microM, and the addition of ZnCl2 led to a concentration-dependent increase in epifluorescence. CdCl2 or PbCl2 at 8 microM was as potent as ZnCl2 at 20-80 microM, whereas NiCl2 at 80 microM was without effect. ZnCl2 completely abolished the inhibition of glycolysis by glucagon (cAMP). Glucagon led to a pronounced drop in cytosolic Zn2+. Both glucagon and zinc stimulated glycogenolysis by increasing the phosphorylation of glycogen phosphorylase but acted oppositely on glycolysis. Zinc overcame the inactivation of pyruvate kinase by glucagon without changing the hormone-induced protein phosphorylation. The antagonistic action of zinc and cAMP on glycolysis together with the rapid and marked decrease in free zinc concentration induced by glucagon (cAMP) may indicate an as yet unknown role of zinc as an important mediator of regulation of carbohydrate metabolism.

The photoreactive AMP analog, g-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the K , for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 pM and 1.5 pM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Since the initial report [ 13 that the biodegradative threonine dehydratase of Escherichia coli, which catalyzes the pyridoxal phosphate-dependent dehydration of threonine and serine to ammonia and corresponding 2-0x0 acids, is stimulated by AMP, considerable evidence has been gathered on the effect of this nucleotide on the conformational states, catalytic mechanism, and regulation of enzyme activity by various cellular metabolites. For example, the enzyme can bind 4 mol AMP/mol protein [2], and the AMP-free enzyme exists as a protomer of M , 35000, whereas the AMP-bound form is a tetramer of M , 140000 which exhibits a 25-fold decrease in the K , for threonine [2-41. The stimulatory effect of AMP has been correlated with obligatory oligomerization of the protomeric species [4]. Furthermore, allosteric inhibition of enzyme activity [5 -71 and catabolite inactivation by covalent protein modification of the dehydratase by intermediary metabolites [7 -91 are known to be precisely regulated by subtle variations in the concentration of AMP. Nevertheless, the binding site for AMP on the enzyme molecule remains unidentified. Recently, the complete amino acid sequence of the E. coli threonine dehydratase has been determined in this laboratory from the nucleotide sequence of the cloned gene [lo, 111, and the active-site pyridoxal-phosphate-bound lysine residue involved in the dehydration reaction has been localized on the protein primary structure [ I l , 121. This report is concerned with photolabeling of the binding site of 8-azido-AMP, a photoaffinity analog of AMP. The experiments revealed that 8-azido-AMP can mimic the allosteric modifier AMP and binds to a unique site on the dehydratase molecule which most likely represents the binding region of AMP.

No abstract

The toxicity and the metabolism dependent irreversible binding of the lipophilic DDT metabolites 3‐methylsulfonyl DDE (MeSO2 DDE) and o,p DDD in the adrenal gland of the chicken were examined As determined by histopathology, vacuolar degeneration and pycnosis of adrenal interrenal cells occurred 4 d after injection of a single dose of either compound (0 25 mmol/kg, 80–100 mg/kg body weight) to 6‐d old chicken In addition, o,p DDD, but not MeSO2 DDE, induced fatty degeneration of the liver According to autoradiography, irreversibly bound radioactivity was present in the interrenal cells after injection of the 14C‐labeled compounds into the air sacs of eggs Both DDT metabolites were also irreversibly bound to protein after incubation with adrenal 300 g supernatant in vitro, with apparent Km values of 2 0 and 8 3 μM for MeSO2 DDE and o,p DDD, respectively The irreversible binding in vitro was inhibited by metyrapone, SKF 525A, and carbon monoxide, suggest ing a cytochrome P450‐dependent metabolic activation to reactive intermediates Addition of glutathione to the incubations reduced the irreversible binding of MeSO2‐[14C]DDE significantly, whereas the irreversible binding of o,p [14C]DDD remained un changed The results indicate that MeSO2‐DDE and o,p DDD are adrenocorticolytic following an adrenal P450‐dependent met abolic activation in the chicken Experiments with embryonated eggs from common eider and domestic duck demonstrated that MeSO2 DDE and o,p DDD are also metabolically activated and irreversibly bound to adrenal tissue in embryos of these species

We have recently described the sequence of the Zn2+-binding domain (43 amino acid residues) of a newly detected Zn 2+-binding protein which reversibly inactivates phosphofructokinase-I in a Zn2+-dependent manner [(1986