Glucogen synthesis in rat liver in vivo was measured by the incorporation of 3H from 3H2O into glycogen. In meal-fed rats incorporation and the incorporation of 3H into glycogen was linear up to 100 min. Before feeding glycogen concentration and the incorporation of 3H were both low; and both rose on feeding to give maximal values after 2-3h. The glycogen concentration was maintained for a further 5h but the incorporation of 3H rapidly declined to pre-feeding values. This shows that glycogen turnover was low in the post-prandial rat. Streptozotocin diabetes decreased the rise in glycogen concentration on feeding and had a similar effect on 3H2O incorporation. Both effects were reversed by insulin administration. The number of 3H atoms incorporated per glycogen glucose moiety formed in biosynthetic experiments (2.84 +/- 0.47) was relatively constant and allowed absolute biosynthetic rates to be calculated. Degradation of glucose from glycogen labelled by 3H2O showed that most of the 3H was located at C-2 and C-5. The incorporation would arise by rapid equilibration of hexose phosphates through phosphoglucose isomerase, transaldolase and triose phosphate isomerase.
Preobese "fatty" rats have been identified by their lower rectal temperature. Of 51 pups born from matings of heterozygote (Fafa) parents, 16 had low rectal temperatures from day 16 onward (34.6 + 0.2 ~ C v 35.4 _+ 0.3 ~ C) and all subsequently became obese. No animal with the higher 'normal' rectal temperature developed obesity. Hepatic fatty acid synthesis (preobese 0.6 _+ 0.1; lean 0.6 _+ 0.1 ~tmol/ g/h), hepatic glucose-6-phosphate dehydrogenase activity (G6PDH) (preobese 0.68 _+ 0.07; lean 0.71 +_ 0.03 ~tmol/g/min) and serum insulin (preobese 64 _+ 2; lean 58 _+ 4 ~tU/ml) were unchanged in 18 day preobese, suckling fafa rats. 3 days after weaning hepatic lipogenesis (preobese 25.3 _+ 2.0; lean 5.4 +_ 0.7 gmol/g/h) and G6PDH activity (preobese 4.5 _+ 0.5; lean 0.90 _ 0.05 ~tmol/g/min) had increased in both lean and preobese rats although the values attained in preobese rats were significantly greater than in lean rats. When weaning was delayed there was no enhancement in lipogenesis, G6PDH or serum insulin in the preobese rat. The results suggest that the primary genetic defect in 'fatty' rats is not related to the increase in lipogenesis or serum insulin but may reflect a defective thermogenic process.
The detailed proof of the 437-residue amino acid sequence (Mr 48,969) of porcine heart citrate synthase (EC 4.13.7) is described. The S-carboxymethylated protein has been cleaved at methionine (cyanogen bromide) and arginine (trypsin digest of citraconylated enzyme) residues to yield 14 and 17 major peptides, respectively. Peptides were initially fractionated by gel filtration, and those useful for sequence analysis were purified by high-performance liquid chromatography. Sequence analyses were performed on these primary peptides and on subpeptides generated by cleavage with the bromine adduct of 2-[(2-nitrophenyl)sulfenyl]-3-methylindole, Staphylococcus aureus V8 protease, trypsin, chymotrypsin, or acid. The overall sequence was confirmed by analyzing products of cleavage by hydroxylamine, acid, and subtilisin. A novel feature of the sequence is the identification of trimethyllysine at residue 368.
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