beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : I. Identification of an Endogenous beta-Linked Glucolipid Activator.

Heat-stable activators of membranous beta-glucan synthase have been isolated from the supernatant fraction of crude mung bean (Vigna radiata) extracts by DEAE-cellulose and silica-gel chromatography. One of the activators has been partially purified and characterized on the basis of susceptibility to various enzymes and by analysis of the products formed upon total acid hydrolysis, alkaline-methanolysis, and beta-glucosidase digestion. This activator has the characteristics of a 1,2-dioleoyl diglyceride containing beta-linked glucose residue(s) at the C-3 position. When expressed per mole of glucosyl residues, the maximal K(a) value of the activator is estimated to be 25 micromolar. Both the intact glucosyl and fatty acid moiety are essential to the stimulatory effect of the activator.

beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : II. Comparison of Effects of an Endogenous beta-Linked Glucolipid with Synthetic n-Alkyl beta-d-Monoglucopyranosides.

n-Alkyl (C(6)-C(12)) beta-d-monoglucopyranosides have been found to be highly potent activators of mung bean beta-glucan synthase in vitro, increasing the V(max) of the enzyme as much as 60-fold and with K(a) values as low as 10 micromolar. Activation is highly specific for the beta-linked terminal glucose residue; other alkyl glycosides such as, octyl-alpha-glucoside, dodecyl beta-maltoside, 6-lauryl sucrose, 6-lauryl glucose, which lack this structure, are ineffective as activators. Based on the similarities in their structure and effects on beta-glucan synthesis under a variety of conditions, it is proposed that the alkyl beta-glucosides are structural analogs of the native glucolipid activator of beta-glucan synthase isolated from mung bean extracts.

Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid.

Cellulose is the most abundant renewable carbon resource on earth and is an indispensable raw material for the wood, paper, and textile industries. A model system to study the mechanism of cellulose biogenesis is the bacterium Acetobacter xylinum which produces pure cellulose as an extracellular product. It was from this organism that in vitro preparations which possessed high levels of cellulose synthase activity were first obtained in both membranous and soluble forms. We recently demonstrated that this activity is subject to a complex multi-component regulatory system, in which the synthase is directly affected by an unusual cyclic nucleotide activator enzymatically formed from GTP, and indirectly by a Ca (2+) -sensitive phosphodiesterase which degrades the activator. The cellulose synthase activator (CSA) has now been identified as bis-(3' 5')-cyclic diguanylic acid (5'G3'p5'G3'p) on the basis of mass spectroscopic data, nuclear magnetic resonance analysis and comparison with chemically synthesized material. We also report here on intermediary steps in the synthesis and degradation of this novel circular dinucleotide, which have been integrated into a model for the regulation of cellulose synthesis.

An unusual guanyl oligonucleotide regulates cellulose synthesis in Acetobacter xylinum.

The mechanism of GTP-specific activation of the membrane-bound cellulose synthase system of Acetobacter xylinum has been further elucidated. The supernatant fraction derived from washed membranes of this organism contains an enzyme which reacts with GTP to form a low molecular mass, heat-stable compound,tentatively characterized as a cyclic oligonuleotide composed of GMP residues, which is the immediate activator of the cellulose synthase. This activation is reversed by a membrane-bound enzyme that degrades the activator; the latter enzyme is inhibited by Ca (2+). It is suggested that the interaction between these enzymes and nucleotide derivatives, mediated by Ca (2+), may regulate cellulose synthesis in VIVO.

Cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, induces cytodifferentiation of follicular granulosa cells cultured in serum-free medium.

Cultured granulosa cells from intact immature rats produced large amounts of progestins in response to phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). MIX alone stimulated up to 9 times higher amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) when compared with FSH (100 ng/ml)-stimulated steroidogenesis. Combined action of MIX and FSH did not yield more activity than MIX action alone. MIX activity was demonstrable exclusively in serum-free culture medium (Dulbecco modified Eagle's medium/F-12) supplemented with insulin, transferrin, hydrocortisone, and fibronectin. Serum severely inhibited MIX induction of 20 alpha-OH-P production. MIX also caused dramatic cell shape changes which occurred within 14 h of exposure to the drug. The cells assumed a spherical shape and remained so as long as MIX was maintained in the culture medium. Upon removal of the drug, the cells respread and acquired the morphology of luteinized cells. In contrast to FSH action, MIX failed to induce the appearance of functional LH receptors. However, similar to FSH effect on immature granulosa cells, MIX successfully induced aromatase enzyme throughout 12 days in culture. MIX alone caused only a minute increase in the accumulation of cAMP. cAMP content in cells induced with FSH (100 ng/ml) was 46 times higher than the cyclic nucleotide levels measured under maximal effective doses of MIX. The discrepancy between the intensive steroidogenic activity of MIX and its inability to substantially raise cAMP content suggests a possible cAMP-independent mechanism for steroid production in the ovarian cells.

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells.

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or alpha-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.