Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Audit of compliance with antenatal protocols.

OBJECTIVE--To assess the implementation of action protocols dictated by antenatal risk factors noted at the initial (booking) antenatal visit. DESIGN--Retrospective study of 2000 women delivered between 1 March 1990 and 29 March 1991. SETTING--Maternity department of a district general hospital supporting a multiethnic population in inner London. MAIN OUTCOME MEASURES--Comparison of clinical actions performed against those dictated by the department''s protocols. Analysis according to clinical importance, gestation at booking, maternal age, parity, birth order, ethnic origin, and certainty of gestational age. RESULTS--Interobserver agreement between the two auditors was good (kappa statistic for risk factors detected, 0.78; for actions generated, 0.80). Of the 15,658 actions dictated by department protocols, 3673 (23.5%) were actually performed by the clinicians. The 63 combinations of risk factors and actions believed by consultants to be of particular clinical importance had an action rate of 28.3% compared with 18.6% for those considered less important (p < 0.001). Mothers who first visited the hospital antenatal clinic at or before 24 weeks'' gestation had 25.2% of relevant protocols fulfilled (p < 0.001). Compliance was significantly improved in women aged 36 or over (32.4%), black women (24.9%), and cases of uncertain gestation (24.5%). Parity and birth order were not associated with an altered action rate. Ethnic origin deemed as "other" (than white, black, Asian, or oriental) or "unknown" was associated with poor compliance (19.3%). CONCLUSIONS--Compliance to a set of agreed protocols was poor even though a computer system was available and a protocol manual had been distributed. Protocols were more likely to be implemented in women who booked early and in some groups of women deemed at high risk including older mothers, black women, and those denoted as having uncertain gestational age.     

Carbonisation of oil palm trunks at moderate temperatures

Thousands of tonnes of dry oil palm trunks will be produced annually in Malaysia after about 1990. A project was initiated to study the feasibility of converting palm trunks into charcoal. Carbonisation was done at terminal temperatures of 400–550°C with holding times of 1–3 h and at two heating rates. From laboratory-scale pyrolysis studies, it was found that holding time does not affect the quantity and quality of the charcoal produced, while heating rate has a minor influence. However, as terminal temperature increases, both yield and volatile content decrease while the fixed carbon content increases. The calorific value and ash contents are independent of the parameters studied and their respective values are 4032 kcal/kg and 37.2%. Since the calorific value is low and the ash content high, it is concluded that oil palm trunks are not suitable for the production of charcoal fuel.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of protein phosphorylation in pancreatic acini by cyclic AMP-mediated secretagogues: interaction with carbamylcholine

The effects on protein phosphorylation in mouse pancreatic acini of cyclic AMP-mediated secretagogues and the Ca2+-mediated agonist carbamylcholine were compared. Under the conditions adopted for the study of protein phosphorylation, carbamylcholine (3 microM) stimulated amylase release from pancreatic acini 6-fold, whereas vasoactive intestinal polypeptide (VIP) (100 nM) and the cyclic AMP analogue 8-bromo-cyclic AMP (1 mM) caused little or no increase in secretion. However, VIP and 8-bromo-cyclic AMP, when added in combination with carbamylcholine, potentiated the stimulation of amylase release to 170-180% of that caused by carbamylcholine alone. As assessed by two-dimensional gel electrophoresis, VIP reproduced four of the ten changes in protein phosphorylation elicited by carbamylcholine, these changes being the increased phosphorylation of one soluble protein and the decreased phosphorylation of three soluble proteins. VIP enhanced the carbamylcholine-induced changes in phosphorylation for three proteins. In addition, VIP increased the phosphorylation of a unique protein of Mr 52,000 and pI 5.66 which was not affected by carbamylcholine. All of the effects on protein phosphorylation exerted by VIP in the presence or absence of carbamylcholine were mimicked by 8-bromo-cyclic AMP. Secretin also reproduced most of the changes in protein phosphorylation caused by VIP, although concentrations of secretin of at least 100-fold higher were required to elicit a maximal response. It is concluded that cyclic AMP-mediated secretagogues alter the phosphorylation of a unique protein as well as of several pancreatic proteins affected by carbamylcholine. Moreover, these effects appear to be mediated primarily by VIP-preferring receptors and may be involved in the synergistic action of VIP to promote carbamylcholine-induced amylase release.     

Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.     

Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.