Increase In Lyase Activity Research Articles

Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4xHis tag is replaced by 3xGly6xHis. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258.

Multiple forms of pectic lyases and polygalacturonase fromFusarium oxysporumf,.sp.radicis lycopersici: regulation of their synthesis by galacturonic acid

The r2 isolate of Fusarium oxysporum f.sp. radicis-lycopersici produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We detected three forms that have lyase activity, four forms with polygalacturonase activity and one form with pectinesterase activity. Lyases had an absolute requirement for calcium and pIs of 9.20, 9.00, and 8.65. The two more alkaline forms had a weak preference for pectin, whereas the other was more active on polygalacturonate. Polygalacturonases had pIs of 9.30, 7.35, 6.85, and 6.55 and were inhibited by calcium ions. Lyases and polygalacturonases were induced by galacturonic acid and were subject to catabolite repression. Induced synthesis occurred at pHs 5.5 and 8.0 and no increase in lyase activities were promoted by alkalinization of cultures. Pectin lyase had an endo mode of action, whereas pectate lyase and polygalacturonase behaved more as exoenzymes. These results are discussed in relation to the appearance of the different pectic enzymes when the fungus is confronted with a pectic polymer.Key words: Fusarium oxysporum f.sp. radicis-lycopersici, Lycopersicon esculentum, pectate lyase, pectin lyase, polygalacturonase.

Modulation of 17α-hydroxylase/C17,20-lyase activity in porcine theca cells

The major source of ovarian androgen is the theca cells. Androgens are produced by the conversion of progestins by the 17α-hydroxylase/C17,20 lyase enzymatic system (lyase). The 3β -hydroxy steroid dehydrogenase and aromatase enzymes in the theca cells are modulated by gonadotropins as well as by steroids produced locally. Therefore, the combined effects of hCG plus progesterone, estradiol, or dihydrotestosterone (DHT) on microsomal lyase activity in theca cells from large and medium-sized follicles were determined. Theca cells (3 × 10 6 cells/6 ml/well) were cultured in Medium 199 (M 199) containing only insulin (10μg/ml) and transferrin (5 μg/ml). At 24 h, theca cells were treated with M 199, hCG (15 ng/ml), progesterone, estradiol, or DHT (100 ng/ml) or a combination of hCG + one of the three steroids. Media were removed at various times of culture (27–72 h) and levels of androgen determined by RIA. Microsomes were incubated with 1μCi [ 3H]progesterone + 0.5mM NADPH and radioactive conversion products were measured after purification by thin layer chromatography. Administration of progesterone, estradiol, or DHT alone had little effect on lyase activity in theca cells from medium-sized follicles whereas the addition of hCG alone significantly increased lyase activity in these cells. However, concomitant addition of any steroid with hCG inhibited the increase in lyase activity after the addition of hCG alone. Theca cells from large porcine follicles had a higher basal level of lyase activity compared to theca cells from the smaller follicles. Lyase activity in theca cells from large follicles was enhanced by progesterone; estradiol was inhibitory. DHT initially stimulated lyase activity in theca cells from large follicles, but was inhibitory later in culture. In contrast to its marked effect on theca cells from medium follicles, hCG had only a small effect on lyase activity in theca cells from large follicles. Thus, thecal lyase activity increased as the follicle matured, providing more androgen substrate for the production of estrogen. Lyase activity in theca cells of medium follicles appears to be regulated predominantly by gonadotropin from the pituitary while intraovarian regulation of lyase activity by steroids may be more important in larger follicles.