Biosynthesis of a glycolipid in starch grains from sweet corn

An enzymic preparation from peas (Pisum satisum), able to fonn netral ad polar glycosides, is described. The best sugar donor is UDPglucose ad the acceptors are present in the enzymic system. of the neutral glucolipid-synthesizing activity, and the fraction that sediments at 100,000g is rich in polar glucolipid glucosyl transferase activity. This latter activity was strongly dependent on Mg+ concentration, the optimum being around 5 to 10 mM. UDP inhibits the reaction and 0.2 to 0.5% (v/v) Triton X-100 has a stimulatory effect. The optimum pH is 7.5.

“…These results are in agreement with those of other authors (7,10,12,17) (12). In the experiments reported here, no evidence was found for acylated glucose.…”

Section: Discussionsupporting

confidence: 94%

Membrane-bound UDP-Glucose

Lezica¹,

Romero²,

Dankert³

1976

“…After the addition of digitonin the synthesis of ASG was not inhibited 100%7b as would be expected if it were produced from free sterol and an acyl-glucose precursor. The exact reciprocal relationship between declining SG and increasing ASG endorses the precursor product relationship proposed on the basis of kinetic data (13). The effect of digitonin was specific for steryl glycoside biosynthesis.…”

Section: Resultsmentioning

confidence: 99%

“…Different effects of cofactors have been reported for these reactions. Kauss (12) found no effect of Zn2 , Mg2", and Co2" at 2 mm, whereas Lavintman and Cardini (13) reported 2-to 3-fold stimulation of incorporation of glucose from UDP-glucose by 10 mM Mn2+ or Mg2+. Axelos and Peaud-Lenoel (1) found the acylation of SG to be inhibited by 5 mM Mg2l, stimulated 20 to 30%o by I mm ATP, and unaffected by CoA either in the presence or absence of ATP.…”

Section: Micromolarmentioning

confidence: 99%

Inhibition of Steryl Glycoside Biosynthesis by Acyl Coenzyme A and by Digitonin

Garcia¹,

Mudd²

1980

Methods. Spinach leaves (150 g) were finely minced with a razor blade and added to 250 ml 0 C homogenizing medium (0.5 M sucrose, 50 mM Tris-HCl, 1 mm EDTA, pH 7.5). The mixture was homogenized in a Waring Blendor with three 2-s bursts. The homogenate was filtered through four layers of cheesecloth. The filtrate was centrifuged at 250g for 2 min and the pellet was discarded. The 250g supernatant was used for the preparation of centrifugal pellets. The isolation of subcellular organelles by differential centrifugation was as described previously (10). The l,OOOg pellet, the 3,000g pellet, and the 20,000g pellet were each suspended in 8 ml 0 C, 0.5 M sucrose in 95 mm Tris-HCl + 1 mM EDTA (pH 7.5), whereas the 88,000g pellet was suspended in 4 ml sucrose-Tris-EDTA solution. Preparation of an acetone powder from the 20,000g pellet was by essentially the same method previously described (10). In some cases, acetone extraction temperatures were 0 C, instead of -15 C; this method tends to give better extraction of lipids.For enzyme assays, 100 mg of acetone powder were suspended in 4 ml of 0 C, 0.1 M Tris-HCl (pH 7.5) and homogenized to uniformity with a Potter-Elvehjem homogenizer. Sterols or fatty acids were added to the dry acetone powder in acetone at room temperature, and the organic solvent was removed under vacuum at room temperature before suspending in buffer.Reaction Mixtures. The standard reaction mixture contained 0.1 M Tris-HCl (pH 7.5), 0.2 mm UDP-glucose (320,000 dpm UDP-[UL-'4Clglucose), enzyme (3 mg protein), and water to make a final volume of 1 ml. The reactions were initiated by the addition of enzyme, and the incubations were for various times at 30 C. The reactions were terminated by the addition of 3 ml methanolchloroform (2:1, v/v). The reaction mixtures then were extracted according to the method of Bligh and Dyer (3).Individual lipids in the chloroform phase were separated and analyzed by Silica Gel G TLC, using chloroform-methanol-acetic acid-water (85:15:3:2, v/v) as the solvent system. The radioactive areas were located by autoradiography. The radioactive areas then were scraped into scintillation vials, 10 ml liquid scintillation fluid (10 g of PPO + 0.6 g of POPOP + 2 liters of toluene) were added to each vial, and the radioactivity of the samples was counted for 10 min in a liquid scintillation counter

“…(8) with slight modifications (2,6). After incubation with the labeled sugar nucleotide, the starch granules were washed by suspending them in a Vor-'Abbreviation: CTAB: cetyltrimethylammonium bromide.…”

Section: Methodsmentioning

confidence: 99%

Effect of Cetyltrimethylammonium Bromide on the Activity of Particulate Starch Synthetase from Potato Tuber

Lavintman

Cardini

1972

Self Cite

The action of some detergents on the incorporation of glucose from uridine diphosphate glucose or adenosine diphosphate glucose into the potato tuber starch grain was studied. It was found that the cationic detergent, cetyltrimethylammonium bromide, produces a rapid binding of both sugar nucleotides to the grain and a great increase in the incorporation of glucose into the polysaccharide. Kinetic constants of starch synthetase are also modified, there being an affinity increase for both sugar nucleotides. Neutral detergents are without effect and anionic detergents are inhibitors.In a previous communication (6) on the synthesis of sterylglucosides from UDP-glucose in amyloplasts of sweet corn endosperm, we have found that the cationic detergent, cetyltrimethylammonium bromide, inhibited that synthesis but increased considerably the incorporation of radioactivity into the grains. We have now tried to investigate the latter effect to see if it was related to an increase in the activity of starch synthetase (UDP-glucose: a-1,4-glucan a-4-glucosyltransferase, EC 2.4.1.21) included inside the grain (4,8,12) or to some modification of the permeability of the granule.