Experimental determination of the phosphorylation state of phenylalanine hydroxylase

A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]Pi incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2+ ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.

Modulation of cell nucleotide levels of isolated kidney tubules

The mechanisms responsible for the large increases of intracellular ATP levels seen after isolated rabbit proximal tubules are treated with exogenous adenine nucleotides were studied. Exogenous ATP was rapidly degraded via adenosine as far as hypoxanthine. Degradation of AMP to adenosine was substantially inhibited by beta-glycerol phosphate. In studies of the ability of individual exogenous purines to increase intracellular ATP levels, single large doses of adenosine were less effective than equimolar doses of exogenous ATP but were substantially more effective than exogenous inosine or hypoxanthine. Exogenous guanine derived compounds increased only cell GTP. Incremental delivery of smaller doses of adenosine to maintain medium levels greater than 5 microM or inhibition of adenosine deaminase with erythro-9-[3-(2-hydroxynonyl)]adenine or 2'-deoxycoformicin enhanced the nucleoside's effectiveness. However, the initial increase of cell ATP was still greater after treatment with exogenous ATP than after adenosine and, in the presence of adenosine deaminase inhibition, larger increases of cell ATP were produced by 50 microM adenosine than by 250 microM adenosine. These observations are most consistent with substrate inhibition of adenosine kinase by adenosine. Furthermore, the adenosine kinase inhibitor, 5-iodotubercidin, prevented the increases of cell ATP resulting from exogenous adenosine or exogenous ATP. These studies demonstrate how the differential uptake and utilization characteristics of nucleosides and bases can fully account for the increases of intracellular nucleotides produced in isolated tubules by exogenous purines.

Inhibition of renal gluconeogenesis and phosphoenolpyruvate carboxykinase activity by 3-mercaptopicolinic acid: studies in rat, guinea pig, dog, rabbit, and man

The inhibition of gluconeogenesis in isolated kidney tubules by 3-mercaptopicolinate, a specific inhibitor of the phosphoenolpyruvate carboxykinase reaction, is species specific. At any given concentration of the inhibitor, the degree of inhibition is in the order rat > guinea pig ≥ man > rabbit ≥ dog. The intracellular distribution of PEPCK activity (as measured in both the forward and backward directions) supports the conclusion that in the intact cell the cytosolic enzyme is preferentially inhibited. Analysis of the inhibition failed to demonstrate any significant differences between the enzymes, regardless of intracellular or animal source. All enzymes exhibited a similar Ki (2–10 μM) for 3-mercaptopicolinate when assayed in the forward direction. The observed preferential cytosolic inhibition could be due to either in situ differences in metallic ions concentration or a restriction in mitochondrial permeability for mercaptopicolinate.