Proportion Of Synthase Research Articles

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of beta-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of beta-1,3-Glc and beta-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 mum, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.

Glycogen Concentration and Regulation of Synthase Activity in Rat Liver in Vivo

The proportion of liver glycogen synthase in the ac tive (R + I) forms is lower in the fed than in the fasted state, This has been attributed to inhibition of synthase phosphatase by the accumulated hepatic glycogen. We observed that after oral administration of glucose or galactose to fasted rats, hepatic synthase R + I activity first increased, as expected, but then decreased to a nadir significantly below the control level regardless of the maximal glycogen concentration reached. Therefore, we investigated further the relationship of hepatic synthase R + I activity and glycogen concentration in vivo in fasted rats given increasing oral glucose loads. Male rats fasted 24 h were given glucose doses of 0.1-4.0 g/kg by oral gavage ( n ≥ 8). Liver synthase R + I, total synthase, phosphorylase a, glucose, glycogen, glucose-6- P, and UDPglucose were measured at intervals over 20-240 min after gavage, Even the smallest glucose load elicited rapid glycogen synthesis. The maximum glycogen concentration increased linearly with the size of the glucose load, although the proportion of administered glucose accounted for by liver glycogen decreased as the glucose load increased. The proportion of synthase in the active (R + I) forms peaked at 20 min after all doses and then declined. By the time the glycogen concentration was maximal, synthase R + I activity had decreased to the control value, regardless of the glucose dose administered or the maximum glycogen concentration reached. Although the decrease in synthase R + I at the time of the glycogen peak was correlated with the increase in glycogen concentration, synthase R + I continued to decrease for another 1-2 h even though liver glycogen was stable or decreasing. The nadir reached was independent of the maximal glycogen concentration. The synthase R + I nadir also did not correlate with hepatic glucose or glucose-g- P concentrations or phosphorylase a activity. Overall, there was not a straightforward temporal or quantitative relationship between the glycogen concentration and synthase R + I activity. These data suggest a more complex mechanism than simply direct inhibition of synthase phosphatase by glycogen.

An improved assay for hepatic glycogen synthase in liver extracts with emphasis on synthase R

An assay for measurement of optimal amounts of glycogen synthase R, the physiologically active form of the enzyme, in liver tissue extracts is described. Tissue extracts enriched in synthase R had a pH profile different from those reported for synthase D and synthase I. In tissue extracts, synthase I had a broad pH optimum but maximal activity was present at pH 7.0–9.0. Synthase D had a sharp pH optimum at pH 8.5 and had little activity at pH 7.0, either in the presence or in the absence of glucose 6-phosphate (G6P). In extracts enriched in synthase R, the pH optimum was 7.0–8.0 without G6P, but 8.0 with G6P. The synthase R activity without G6P rapidly decreased at a higher pH. The proportion of synthase in the physiologically active form traditionally has been reported as an activity ratio based upon the activity in the presence and absence of G6P. The assay has been performed at a single pH. Because of the differences in pH profile, we recommend that the enzyme be measured at pH 7.0 in the absence of G6P and pH 8.5–8.8 in the presence of G6P. In previous assays the substrate UDP-Glc concentration used often has been less than saturating, and the G6P concentration generally has been excessive. A substrate concentration of 11 m m UDP-Glc was found to be necessary for maximal activity. A G6P concentration of 2 m m is adequate for measurement of the D form of the enzyme.

Acute effects of ingestion of carbohydrate, protein, or fat on cardiac glycogen metabolism in rats

Administration of large doses of insulin to intact rats has been shown to stimulate cardiac glycogen synthase phosphatase activity. This results in an activation of glycogen synthase. However, whether a more physiologic stimulus for insulin secretion also resulted in activation of synthase had not been studied. In the present study, rats were fed glucose, casein hydrolysate, or a mixed meal after a 24-hour fast as a means of physiologically stimulating insulin secretion. Lard was fed as a noninsulinotropic nutrient for comparative purposes. Plasma insulin was significantly increased by 15 minutes in rats given a mixed meal or glucose, but surprisingly, no change was observed in rats fed casein hydrolysate. As expected, no change in plasma insulin was observed in rats fed lard. Synthase phosphatase activity was stimulated in rats fed a mixed meal, glucose, or casein hydrolysate, but not in rats fed lard. Likewise, the proportion of synthase in the active (I) form was significantly increased in rats fed a mixed meal, glucose, or casein hydrolysate, but not in rats fed lard. The increase in phosphatase activity and the increased proportion of synthase in the active form following ingestion of casein hydrolysate was unexpected since this occurred in the absence of an increase in insulin. The proportion of phosphorylase in the active ( a) form decreased in rats fed glucose, but remained unchanged in rats fed a mixed meal, casein hydrolysate, or lard. The cardiac glycogen concentration decreased dramatically in rats fed casein hydrolysate. Following the other meals, there was either no change or the decrease was modest. Thus, casein hydrolysate ingestion caused a decrease in cardiac glycogen in the absence of a change in the proportion of phosphorylase in the active ( a) form, and in the presence of an increase in the proportion of synthase in the active (I) form. The mechanisms involved in these casein hydrolysate stimulated events are unknown.

Synthase activation is not a prerequisite for glycogen synthesis in the starved liver.

To evaluate the contribution of phosphorylase and synthase interconversion as well as the availability of substrates to the onset of liver glycogen synthesis, this process was studied in rats starved overnight and refed for 4 h. On feeding, phosphorylase kinase and phosphorylase were inactivated in a cAMP-independent way, but the proportion of synthase a was unchanged and associated with increased hexoses 6-phosphate (glucose and fructose 6-phosphate), uridine diphosphoglucose (UDPG), and fructose 2,6-bisphosphate concentrations. These findings serve to support a "push" mechanism whereby substrate availability for synthase a concerted with phosphorylase inactivation provokes glycogen deposition. Anesthesia was compulsory for liver sampling and analysis. If such experiments were carried out in conscious rats killed by decapitation, artefactual cAMP-dependent phosphorylase activation and synthase inactivation were observed in starved animals. The phosphorylase activation persisted in refed animals but by a cAMP-independent mechanism.

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active ( a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in the a form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg ++ ratio or by an increase in intracellular Ca ++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.