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Transfection experiments using Xenopus vitellogenin A2 gene constructs allowed us to identify an activator which increases the activity of the thymidine kinase promoter. The activator is located between −121 and −87 of the A2 vitellogenin gene and is separated by a stretch of curved DNA from the estrogen‐responsive DNA element at −331. The activator functions in a cell‐specific manner, as it is active in human breast cancer cells (MCF‐7) as well as hepatoma cells but not in fibroblasts or HeLa cells. The activator is composed of at least three elements: elements 1 and 2 which form a partial palindrome, function independently, but act synergistically when combined. Element 3 is not active on its own, but supports elements 1 and 2. A TATA box region derived from the Xenopus albumin gene is sufficient for the function of the activator. In vitro transcription experiments using rat liver nuclear extracts demonstrate that the activator interacts with transcription factors. These factors are distinct from those recognizing HP1, a regulatory element common to several genes specifically expressed in hepatocytes.

The enzyme system from Clostridium sticklandii that catalyzes the migration of the 6-amino group of D-alysine to carbon 5, forming 2,5-diaminohexanoate, has been purified to near homogeneity as a complex of two dissimilar proteins, a red cobamide protein (Ei) and a sulfhydryl protein (E2). The complex is dissociated by acidification to pH 4.0; the precipitate which forms contains Ei while E2 remains in the supernatant solution. The combined fractions are catalytically active. The activity of the isolated D-a-lysine mutase complex depends on added pyridoxal phosphate, Bi2-coenzyme, and a mercaptan. Mutase activity is stimulated by ATP, Mg2+, and a monovalent cation such as K+ or NH4+. ATP, which can be replaced by its phosphonic acid analogs, is an allon the basis of early isotope studies it was concluded that the fermentation of DL-lysine to acetate, butyrate, and ammonia by Clostridium sticklandii involves two separate pathways (Stadtman and White, 1954;. In one pathway, which could be investigated in soluble extracts (Stadtman, 1963), acetate is formed from carbon atoms 1 and 2 of lysine and butyrate is formed from the remainder of the molecule. The primary substrate, L-a-lysine, first undergoes two successive amino group migration reactions (Chirpich et al., 1970;Tsai and Stadtman, 1968;Stadtman and Renz, 1968) and then is oxidatively deaminated to form 3-keto-5aminohexanoate (Rimerman and Barker, 1968) before eventual cleavage of the carbon chain occurs (Figure 1).In the other pathway acetate is formed from carbons 5 and 6 of lysine and butyrate is derived from the carboxyl end of the molecule. Soluble extracts fail to carry out this overall series of reactions and, therefore, the reaction sequence is still unknown. However, the recently discovered conversion of D-a-lysine into 2,5-diaminohexanoate (Stadtman and Tsai, 1967) presumably is the first step. (Figure 1). In both lysine pathways there is a Bi2-coenzyme-mediated migration of the e-amino group to carbon 5; in the L-lysine pathway this is the second enzymic step and the product is 3,5-diaminohexanoate whereas in the D-lysine pathway it is the first step and the product is 2,5-diaminohexanoate. The two Bi2-coenzyme-• From the

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A B S T R A C T Total plasma immunoreactive pancreatic glucagon (IRG) was measured in samples taken simultaneously from the proximal portal vein and superior vena cava of 26 healthy rats. The portalperipheral ratio of IRG was 2.80±0.25, the portalperipheral difference (A) 124±15 pg/ml, and percentage extraction 58±+3. Gel filtration of paired portal and peripheral vein samples showed that reduction in the 3,500-dalton IRG component (glucagon) in peripheral samples accounted for almost all the differences, there being minimal and inconsistent changes in the high molecular weight (>40,000) fraction. The portalperipheral ratio of the 3,500-dalton glucagon was 5.24±+1.10, the portal-peripheral difference 130+33 pg/ml, and the percentage extraction 81±5.To study the transhepatic differences in the 9,000-dalton "proglucagon-like" material, the experiment was repeated in nine rats 24 h after bilateral nephrectomy, a procedure which increases plasma levels of this fraction. The portal-peripheral ratio for plasma IRG in these rats was 1.48+0.12, the portal-peripheral difference 140±29 pg/ml, and percentage extraction 28+5. Gel filtration revealed no consistent differences between portal and peripheral concentrations of the 9,000-and >40,000-dalton components, which comprised 40 and 13%, respectively, of the mean IRG level of 492+35 pg/ml. In contrast, there were marked differences between portal and peripheral levels of the 3,500-dalton component, the ratio being 3.42 +0.63, the portal-peripheral difference 182+32 pg/ml, and percentage extraction 64±5. Similar studies in a healthy dog, in which species there are significant circulating levels of the 9,000-dalton IRG component, confirmed the selective hepatic extraction of the 3,500-dalton fraction. We conclude that the various IRG fractions are metabolized differently by the liver, and that portalperipheral ratios based on direct assay of plasma IRG will vary depending on the percentage glucagon immunoreactivity in each fraction; the greater the combined contribution of fractions other than the 3,500-dalton component to total plasma IRG, the lower will be the ratio. Because of the heterogeneity of circulating IRG and significant differences in the metabolism of its various components, gel filtration of plasma samples is necessary for precise quantitation of the hepatic uptake of each particular fraction.