The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

A simple kinetic test to distinguish exo-glucosidase and beta-glucosidase activities

Unusual kinetic behaviour was observed in assaying spectrophotometrically for exo-glucanase activity in a beta-glucosidase isolated from A. faecalis using p-nitrophenyl beta-cellobioside as substrate. At high substrate concentrations no phenol was released whereas at low concentrations a rapid release of phenol was detected and this increased in rate with extent of hydrolysis. These results are consistent with a model involving tight binding of the substrate to the enzyme and an initial exo-glucosidase-catalysed hydrolysis to produce glucose and p-nitrophenyl glucoside. Subsequent hydrolysis of the nitrophenyl glucoside results in phenol release, but only after sufficient concentrations have accumulated to compete with the cellobioside. This theory was confirmed by product analysis and by measuring the affinity of the substrate for the enzyme by its inhibition of p-nitrophenyl glucoside hydrolysis. Observation of such kinetic behaviour allows distinction between beta-glucosidase and exo-glucosidase activities.     

Factors affecting the formation of polysaccharide depolymerase and glycoside hydrolyase enzymes by Butyrivibrio fibrisolvens NCDO 2249

Specific activities of hemicellulose-degrading polysaccharide depolymerase and glycoside hydrolase enzymes were measured in batch and continuous cultures of Butyrivibrio fibrisolvens NCDO 2249 grown on cellobiose or a hemicellulosic carbohydrate. Enzyme activities were influenced by the growth substrate and by the rate and stage of growth of the micro-organism. In cellobiose batch cultures specific activities were maximal as the growth rate declined and in the initial stages of the stationary phase. The growth substrate did not affect the range of glycoside hydrolases formed, although specific activities were substrate-dependent, with activity increases (up to 200-fold) occurring in enzymes essential for effective substrate utilization. Appreciable xylanase activity was present only in xylan-grown cultures. The substrate effects were also evident in chemostat cultures. The activity response of the nine enzymes monitored to growth rate changes differed in that while the activity of some enzymes, including xylanase, declined at high dilution rates the activities of others were not growth rate-dependent and were maintained over the range of dilution rates examined. Exocellular activities were detected only in spent media from cultures grown with a polymeric (hemicellulosic) carbohydrate.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

A solid-state 31P-NMR investigation of the allosteric transition in glycogen phosphorylase b.

The catalytic role of the cofactor phosphate moiety at the active site of glycogen phosphorylase has been the subject of many investigations including solution-state high-resolution 31P-NMR studies. In this study the pyridoxal phosphate moiety in both the inactive and active forms of microcrystalline phosphorylase b has been investigated by high-resolution 31P magic-angle spinning NMR. The symmetry of the shielding tensor in model compounds at varying degrees of ionization is investigated and the results indicate a marked difference between the dianionic and monoanionic model compounds. Consequently the observed similarity in the principal tensor components describing the shielding tensor of the phosphorus nuclei present at the active site of both the R- and T-state conformations suggests that there is no change in ionization site upon activation in contrast to suggestions based upon isotropic shifts. Since previous relaxation measurements have pointed to the need to consider motional influences in such systems, several plausible models are considered. Subject to the assumption of congruency between the principal axis system describing the shielding interaction and molecular frame determined by the molecular symmetry axes, we conclude that the phosphate cofactor is dianionic in both forms.     

Isolation and Characterization of Carotenoid-rich Lipid Globules from Peridinium foliaceum

Carotenoid-rich oil globules were isolated from the cytoplasm of the binucleate dinoflagellate, Peridinium foliaceum. These orange globules were collected from ruptured cells by ultracentrifugation on a sucrose density gradient, and checked for purity by electron microscopy. The osmiophilic globules were assayed for lipid (including pigment) and protein content. The lipid to protein ratio was 1.39:1, with a calculated density of the globules of 1.05 grams per cubic centimeter. The lipids were composed of hydrocarbon, wax ester (phytyl ester), triglyceride, and polar (no phospholipid) fractions. The biochemical composition indicated that the globules function as a reservoir of energy-rich components in the cell. Microspectrophotometric observations were consistent with pigment analyses which demonstrated that the globules were carotenoid-rich. In addition to β-carotene, γ-carotene, and canthaxanthin, the carotenogenic precursors: phytoene, phytofluence, ζ-carotene and β-zeacarotene were isolated from the globules. Corrected fluorescence maxima of phytoene and phytofluene in hexane were recorded at 340 and 490 nanometers, respectively. Carotenes constituted 3.3% of the total oil globule lipid. The possibility of an extraplastidic carotenogenic enzyme system in P. foliaceum is discussed.     

A freeze-preservation of synchronously dividing cultured cells of Acer pseudoplatanus L

A suspension culture of sycamore (Acer pseudoplatanus L.) was synchronised in division by release from nitrogen starvation. Cell samples, taken during the lag phase and synchronous growth, were examined cytologically and subjected to a freeze-preservation protocol. A high positive correlation was found between mitotic index (percentage of cells showing mitotic figures) and cell survival, as measured by fluorescein diacetate staining and reduction of 2,3,5-triphenyl tetrazolium chloride. Specific staining of lethally damaged cells and subsequent examination of the surviving cells demonstrated that the latter had a lower mitotic index and more consistent, low value of nuclear DNA than the total population. This indicated that it is cells, newly entered into G₁ phase of the cell cycle, which are particularly resistant to the stresses imposed by the freeze preservation protocol.