Biosynthesis of KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). Identification and characterization of a KDN-9-phosphate synthetase activity from trout testis.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Synthesis of methyl alpha-D-glucooligosaccharides by entrapped dextransucrase from Leuconostoc mesenteroides B-1299

The synthesis of methyl alpha-D-glucooligosaccharides, using sucrose as glucosyl donor and methyl alpha-D-glucopyranoside as acceptor, was studied with dextransucrase from Leuconostoc mesenteroides NRRL B-1299. The enzyme was immobilized by entrapment in alginate. By NMR and mass spectrometry we identified three homologous series (S1-S3) of methyl alpha-D-glucooligosaccharides. Series S2 and S3 were characterized by the presence of alpha(1-->2) linkages, in combination with alpha(1-->6) bonds. Two parameters, sucrose to acceptor concentration ratio (S/A) and the total sugar concentration (TSC) determined the yield of methyl alpha-D-glucooligosaccharides. The maximum concentration achieved of the first acceptor product, methyl alpha-D-isomaltoside, was 65 mM using a S/A 1:4 and a TSC of 336 g l(-1). When increasing temperature, a shift of selectivity towards compounds containing alpha(1-->2) bonds was observed. The formation of leucrose as a side process was very significant (reaching values of 32 g l(-1)) at high sucrose concentrations.     

Structure of the O-specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33.

The acidic O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H-NMR and 13C-NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain D-ribitol 5-phosphate (D-Rib-ol-5-P) and ethanolamine phosphate (Etn-P) and has the following structure: D-Rib-ol-5-P (3) approximately 75% EtnP(6)-->2)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-GlcpNAc-(1-->). This structure is identical with that of the O-polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O-antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross-reactivity of P. mirabilis D52 O-antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O-polysaccharides. The role of charged groups, Rib-ol-5-P and Etn-P in the immunospecificity is discussed.     

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides

For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides. All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside. In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst. The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for alpha-butylglucopyranoside and alpha-octylglucopyranoside, respectively. Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of alpha-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.     

Enzymic alpha-galactosylation of a cyclic glucotetrasaccharide derived from alternan

Alternanase catalyzes the hydrolysis of alternan, an alpha-(1-->3)-alpha-(1-->6)-D-glucan produced by Leuconostoc mesenteroides, resulting in the formation of a cyclic tetramer cyclo -->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->(2) (cGlc(4)). Two alpha-galactosidases, one from coffee bean and the other produced by a fungus, currently described as Thermomyces lanuginosus, were found to catalyze an efficient 6-O-alpha-D-galactopyranosylation of cGlc(4). The attachment of a nonreducing alpha-D-galactopyranosyl residue to the cGlc(4) molecule opens new possibilities for future applications of the cyclic tetramer, since the D-galactopyranosyl residue can be easily modified by D-galactose oxidase to introduce a reactive aldehyde group. The results also extend our knowledge about the synthetic potential of T. lanuginosus alpha-galactosidase.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

The synthesis of mannose 1-phosphate in brain

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.     

Effects of different levels of N- and P-deficiency on cell yield,okadaic acid,DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate Prorocentrum lima

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N) and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1) contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient conditions (f/2 medium; NO₃⁻ and PO₄³⁻ concentrations: 883 and 36.3 μM, respectively; N/P ratio: 24). Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the pattern observed was dependent on nutrient dynamics, as shown by N and P analyses performed in culture media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50-P-limited levels with respect to control values, while cell volume increased with respect to control (up to 30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to 15.80 pg cell⁻¹, while DTX-1 ranged from 0.12 and 0.39 pg cell⁻¹ resulting in unusual high OA/DTX-1 ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as: (i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentration under P-limitation; (ii) significant increase of cell carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii) significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological significance of this increase is discussed.     

Euglycemic hyperinsulinemia increases glucose 1,6-bisphosphate in human skeletal muscle

1. The effects of physiologic concentrations of insulin on the contents of glucose 1,6-bisphosphate (glucose 1,6-P2) and regulators of glucose 1,6-P2 synthase in intact human skeletal muscle have been investigated. 2. Insulin increased glucose 1,6-P2 from a basal value of 70 +/- 6 to 135 +/- 12 mumol/kg dry wt (P less than 0.001). 3. Activation of synthase could not be associated with changes in its inhibitors (fructose 1,6-P2, Pi, citrate) or its substrate glucose 6-P.     

The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin

Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> [Fe(2)-P](Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and [Fe(2)-P](Z) represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe(2)-P](Z) + (1)/(2)O(2) --> [Fe(2)O-P](Z) where [Fe(2)O-P](Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+). Iron oxidation is inhibited by the presence of Zn(2+) ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center.     

Peptide synthesis catalyzed by subtilisin-72 in organic solvents

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.     

ENZYMIC AND CEREBRAL METABOLIC EFFECTS OF 2-DEOXY-d-GLUCOSE

—The time course of effects of 2-deoxy-d -glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d -glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d -glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d -glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d -glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d -[U-¹⁴C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d -glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d -glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d -glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d -glucose and its conversion to 2-deoxy-d -glucose-6-phosphate, the 2-deoxy-d -glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min. Measurement of enzyme activities in cerebral homogenates (using 1 mivs substrate concentration) showed that hexokinase (EC 2.7.1.1) was 40 per cent inhibited by 5 mm -deoxy-d -glucose (but not by 2-deoxy-d -glucose 6-P). Glucose 6-P dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and phosphoglucomutase (EC 2.7.5.1) were not affected by either 2-deoxy-d -glucose (5 mm ) or 2-deoxy-d -glucose 6-P (5 or 20 mm ). Hexose-phosphate isomerase (EC 5.3.1.9) was 70 per cent inhibited by 20 mm -d -deoxy-d -glucose 6-P. Phosphofructokinase (EC 2.7.1.11) was inhibited by 17 per cent by 2-deoxy-d -glucose 6-P (20 mm ). During the initial impairment of cerebral function by 2-deoxy-d -glucose there is competitive inhibition of glucose transport into the brain; later, glycolysis is more powerfully depressed by the inhibition of isomerase produced by the high intracerebral concentration of 2-deoxyglucose-6-phosphate.     

Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalyzed by activated protein C

Activated Protein C (APC) inactivates factor VIIIa by cleavage at Arg(336) and Arg(562) within the A1 and A2 subunits, respectively, with reaction at the former site occurring at a rate approximately 25-fold faster than the latter. Recombinant factor VIII variants possessing mutations within the P4-P3' sequences were used to determine the contributions of these residues to the disparate cleavage rates at the two P1 sites. Specific activity values for 336(P4-P3')562, 336(P4-P2)562, and 336(P1'-P3')562 mutants, where indicated residues surrounding the Arg(336) site were replaced with those surrounding Arg(562), were similar to wild type (WT) factor VIII; whereas 562(P4-P3')336 and 562(P4-P2)336 mutants showed specific activity values <1% the WT value. Inactivation rates for the 336 site mutants were reduced approximately 6-11-fold compared with WT factor VIIIa, and approached values attributed to cleavage at Arg(562). Cleavage rates at Arg(336) were reduced approximately 100-fold for 336(P4-P3')562, and approximately 9-16-fold for 336(P4-P2)562 and 336(P1'-P3')562 mutants. Inhibition kinetics revealed similar affinities of APC for WT factor VIIIa and 336(P4-P3')562 variant. Alternatively, the 562(P4-P3')336 variant showed a modest increase in cleavage rate ( approximately 4-fold) at Arg(562) compared with WT, whereas these rates were increased by approximately 27- and 6-fold for 562(P4-P3')336 and 562(P4-P2)336, respectively, using the factor VIII procofactor form as substrate. Thus the P4-P3' residues surrounding Arg(336) and Arg(562) make significant contributions to proteolysis rates at each site, apparently independent of binding affinity. Efficient cleavage at Arg(336) by APC is attributed to favorable P4-P3' residues at this site, whereas cleavage at Arg(562) can be accelerated following replacement with more optimal P4-P3' residues.     

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.     

Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase

Cellobiose was tested as acceptor in the reaction catalyzed by alternansucrase (EC 2.4.1.140) from Leuconostoc mesenteroides NRRL B-23192. The oligosaccharides synthesized were compared to those obtained with dextransucrase from L. mesenteroides NRRL B-512F. With alternansucrase and dextransucrase, overall oligosaccharide synthesis yield reached 30 and 14%, respectively, showing that alternansucrase is more efficient than dextransucrase for cellobiose glucosylation. Interestingly, alternansucrase produced a series of oligosaccharides from cellobiose. Their structure was determined by mass spectrometry and [13C-1H] NMR spectroscopy. Two trisaccharides are first produced: alpha-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-D-glucopyranose (compound A) and alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->4)-D-glucopyranose (compound B). Then, compound B can in turn be glucosylated leading to the synthesis of a tetrasaccharide with an additional alpha-(1-->6) linkage at the non-reducing end (compound D). The presence of the alpha-(1-->3) linkage occurred only in the pentasaccharides (compounds C1 and C2) formed from tetrasaccharide D. Compounds B, C1, C2 and D were never described before. They were produced efficiently only by alternansucrase. Their presence emphasizes the difference existing in the acceptor reaction selectivity of the various glucansucrases.     

Multiple Forms of Phosphatase from Human Brain: Isolation and Partial Characterization of Affi-Gel Blue Nonbinding Phosphatase Activities

Phosphatases extracted from a human brain were resolved into two main groups, namely affi-gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue binding phosphatases were further separated into four different phosphatase activities, designated P1-P4, and described previously (1). In the present study we describe the affi-gel blue-nonbinding phosphatases which were separated into seven different phosphatase activities, designated P5-P11 by poly-(L-lysine)-agarose and aminohexyl Sepharose 4B chromatographies. These seven phosphatase activities were active toward nonprotein phosphoester. P7-P11 and to some extent P5 could also dephosphorylate a phosphoprotein. They displayed different enzyme kinetics. On the basis of activity peak, the apparent molecular mass as estimated by Sephadex G-200 column chromatography for P5 was 49 kDa; P6, 32 kDa; P7, 150 kDa; P8, 250 kDa; P9, 165 kDa; P10, 90 kDa and P11, 165 kDa. Immunoblot analysis indicated that P8-P11 may belong to PP2B family, whereas P7 may associate with PP2A. The phosphatases P7-P11 were found to be effective in the dephosphorylation of Alzheimer's disease abnormally hyperphosphorylated tau. The resulting dephosphorylated tau regained its activity in promoting the microtubule assembly, suggesting that P7-P11 might regulate the phosphorylation of tau protein in the brain.