beta-agarases I and II from Pseudomonas atlantica. Purifications and some properties

The agarose-degrading system of Pseudomonas atlantica has been re-examined. In addition to the previously reported extracellular endo-beta-agarase [Yaphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333-335] a second, membrane-bound endo-enzyme activity, beta-agarase II has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose] and also to degrade partially 6-O-methylated agarose to neoagarobiose and 6(1)-O-methyl-neoagarbiose. Novel assays were devised for beta-agarase II and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of beta-agarase I and II. beta-Agarase I was purified 670-fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G-100. The enzyme was resolved from the small amount of extracellular beta-agarase II. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32000. Activity was observed against agar over the pH range 3.0-9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30 degrees C but only for up to 2 h at 40 degrees C. beta-Agarase II was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-100, hydroxyapatite and DEAE-Sepharose CL-6B. This preparation was free of beta-agarase I and disaccharidase. beta-Agarase II was stimulated by NaCl, optimally in the range 0.10-0.20 mol dm-3 (2.4-fold the activity at 0.010 mol dm-3 NaCl). Alkali earth metal (0.002 mol dm-3 CaCl2 or 0.005 mol dm-3 MgCl2) gave 1.2-fold the normal activity. Optimum activity was over pH 6.5-7.5.     

Selective depolymerisation of dermatan sulfate: production of radiolabelled substrates for alpha-L-iduronidase, sulfoiduronate sulfatase, and beta-D-glucuronidase

Radiolabelled disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of disaccharides was approximately 87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total disaccharide fraction. The other disaccharides were O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for alpha-L-iduronidase to produce 2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from alpha-L-iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]talitol residues is an important structural determinant in the mechanism of action of alpha-L-iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from alpha-L-iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.     

A simple method of preparing diverse neoagaro-oligosaccharides with beta-agarase

In order to prepare pure and well-defined oligosaccharides from agarose in a rapid and simple manner, an enzymatic degradation method was developed, which includes degradation with either recombinant beta-agarase (EC 3.2.1.81) AgaA or AgaB and gel permeation chromatography. Agarose was degraded with AgaA at the optimized conditions, yielding 47% and 45% of neoagarotetraose and neoagarohexaose, respectively. These neoagaro-oligosaccharides were conveniently separated by consecutive column chromatography on Bio-Gel P2 or P6 and were identified by FACE. The structure of these neoagaro-oligosaccharides was confirmed by MALDI-TOF MS and (13)C NMR spectroscopy.     

Characterization of the agarase system of a multiple carbohydrate degrading marine bacterium

A marine bacterium strain 2-40 (2-40) degraded numerous complex carbohydrates, such as agar, chitin and alginate. It may play an important role in altering carbon fluxes in marine environments. End-product analyses revealed that 2-40 synthesized an agarase system that consisted of at least three enzymes, beta-agarase I, beta-agarase II and alpha-agarase, which acted in concert to degrade polymeric agar to D-galactose and 3,6-anhydro-L-galactose. The agarase system was shown to be both cell envelope-associated and extracellular, with the relative concentrations depending on the growth phase. The principal depolymerase, a beta-agarase I, hydrolysed agar to both neoagarotetrose and neoagarobiose, as identified by thin layer chromatography. This agarase had a mass of 98 kD and a Pi of 4.3. The agarase system was repressed by D-glucose and D-galactose and induced by agar, agarose, neoagarobiose, neoagarotetrose and neoagarohexose.     

Enzymatic hydrolysis of agar: purification and characterization of beta-neoagarotetraose hydrolase from Pseudomonas atlantica.

Agarose is degraded by a beta-agarase from Pseudomonas atlantica to neoagarooligosaccharides of degree of polymerization (DP), 4, 6, 8, and 10. A beta-neoagarotetraose hydrolase cleaves the central beta-linkage in neoagarotetraose and the beta-linkage near the nonreducing end in neoagarohexaose and -octaose to yield neoagarobiose. The beta-neoagarotetraose hydrolase was localized on or outside the cytoplasmic membrane, in the cell wall region. The enzyme was activated by NaCl, KCl, CaCl2, MnCl2, and MgSO4, has a Km of 3.4 X 10(-3) M for neoagarotetraose, was free from beta-agarase and alpha-neoagarobiose hydrolase activity, and showed no transglycosidic activity.     

STRUCTURE-ACTIVITY RELATIONSHIPS OF OLIGOAGAR ELICITORS TOWARD GRACILARIA CONFERTA (RHODOPHYTA)

Agar oligosaccharides in the neoagarobiose series were prepared by partial enzyme hydrolysis, separated on Biogel P2 and P4, and analyzed by high-performance anion exchange chromatography with pulsed amperometric detection, yielding neoagarosaccharide fractions with a disaccharide repetition degree ranging from 1 (neoagarobiose) to more than 8 (neoagarohexadecaose). These fractions were analyzed for their biological activity toward the marine red alga Gracilaria conferta (Schousboe ex Montagne) J. et G . Feldmann in terms of increase of oxygen consumption, release of hydrogen peroxide, elimination of epiphytic bacteria, and induction of thallus tip bleaching. The structure–activity and dose–response relationships of neoagarosaccharides were very similar in the respiratory and oxidative burst responses and in their bactericidal properties, with neoagarosaccharides consisting of 6 to 8 disaccharide repeating units being the most active. All these responses were competitively inhibited by the reduced form of neoagarohexaose, neoagarohexaitol. In contrast, the tip-bleaching response was light dependent, required much higher concentrations of neoagarosaccharides, and was not inhibited by neoagarohexaitol, suggesting that it is an unspecific oxidative stress reaction. Putative structural effects on the recognition of endogenous agar-oligosaccharide elicitors by G. conferta are discussed.     

OLIGOAGARS ELICIT A PHYSIOLOGICAL RESPONSE IN GRACILARIA CONFERTA (RHODOPHYTA)

Gracilaria conferta (Schousboe ex Montagne) J. et G. Feldmann responded with an oxidative burst and rapid increases in respiration and halogenating activity when agar, agarose, or the agarose degradation products neoagarotetraose and neoagarohexaose were added to the growth medium. In contrast, carrageenan, oligocarrageenans, neoagarobiose, l-galactose, d-galactose, and several other mono- and oligosaccharides did not have any effect. Sixfold increases in respiration were observed 3 min after addition of neoagarohexaose. The response could only be induced in species of the genera Gracilaria and Gracilariopsis. Neoagarohexaose also elicited a release of hydrogen peroxide in less than 15 min, resulting in an immediate increase in algal brominating activity. Bleached thallus tips appeared a few hours after the addition of neoagarohexaose. This effect was dependent on the release of hydrogen peroxide and exposure to light. Exposure to light and oligosaccharide elicitors increased the production of reactive oxygen species, which reached destructive concentrations when both mechanisms were simultaneously active. Concentrations of 0.1 to 3.3 μM agarose or agars were sufficient to trigger an increase in respiration, an oxidative burst response, and tip bleaching. However, higher concentrations of neoagarohexaose and neoagarotetraose were necessary to elicit the responses, indicating that the alga is more sensitive to oligoagars with degrees of biose-polymerization > 3. The extremely short reaction time and high specificity indicate that intermediates of agar degradation are recognized by Gracilaria as messengers when microbial degradation of its cell wall occurs. The physiological responses may represent the early stages of algal defense mechanisms involved in repression of pathogen ingress.     

Preparation from keratin sulfate of substrates for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase and (1 goes to 3)-N-acetyl-beta-D-glucosaminidase

An extract of bacterial cells Pseudomonas sp. IFO-13309 grown on medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-beta-D-gluco-pyranosyl 6-sulfate-(1 goes to 3)-D-galactose, isolated by gel filtration on Sephadex G-25 and purified by preparative paper chromatography. This was reduced with sodium borotritide to give 2-acetamido-2-deoxy-beta-D-glucopyranosyl 6-sulfate-(1 goes to 3)-D-[1-3H]galactitol, purified by gel filtration on Sephadex G-15, which was an excellent substrate for the measurement of 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase. The reduced, radioactive monosulfated disaccharide was desulfated with methanolic 70mM hydrogen chloride and purified by gel filtration on Sephadex G-15 to give O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 goes to 3)-D-[1-3H]galactitol, which allowed the measurement of (1 goes to 3)-N-acetyl-beta-D-glucosaminidase. This enzyme may participate in the normal degradation of keratan sulfate.     

Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.     

Identification and biochemical characterization of Sco3487 from Streptomyces coelicolor A3(2), an exo- and endo-type β-agarase-producing neoagarobiose

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.     

Characterization and in vivo production of three glycolipids from Candida Bogoriensis: 13-glucopyranosylglucopyranosyloxydocosanoic acid and its mono- and diacetylated derivatives

Three glycosides of 13-hydroxydocosanoic acid isolated from Candida bogoriensis were characterized by quantitating the amount of carbohydrate, acetate, and hydroxy acid in each, and by gas-liquid chromatography and mass spectrometry of their methyl ester, trimethylsilyl ether derivatives. One of the glycosides was the diacetylated derivative of 13-glucosylglucosyloxydocosanoic acid previously characterized by Tulloch, Spencer, and Deinema (Can. J. Chem., 46: 345 [1968]), in which the disaccharide had the beta(1 --> 2) sophorose linkage and the acetyl groups were attached to the 6' and 6" positions of the glucose residues. The other two glycosides were 13-glucosylglucosyloxydocosanoic acid and its monoacetylated derivative. A comparison of the mass spectra of derivatives indicates that the acetyl group of the monoacetyl lipid is on the internal glucose. Methyl 13-glucosyloxydocosanoate was produced by acid hydrolysis of the methyl ester of the unacetylated glycolipid and was characterized by the same techniques as the other glycolipids. Time course of production of the three glycolipids is consistent with the diacetylated derivative being the first extra-cellular product and the other two glycolipids being formed by deacetylation. 13-Hydroxy[13-(3)H]docosanoic acid, methyl 13-hydroxy[13-(3)H]docosanoate, and 9-hydroxy[11,12-(3)H]-stearic acid were each incorporated into the glycolipid fraction.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Parasite glycoconjugates. Part 16: Synthesis of a disaccharide and phosphorylated di- and tri-saccharides from Leishmania lipophosphoglycan

A neutral disaccharide beta-D-Galp-(1-->4)-alpha-D-Manp and phosphorylated di- and tri-saccharides beta-D-Galp-(1-->3)-[H(2)PO(3)-6]-beta-D-Galp-O[CH(2)](8)CHCH(2) and beta-D-Galp-(1-->3)-[H(2)PO(3)-6]-beta-D-Galp-(1-->4)-alpha-D-Manp, which are fragments of the phosphoglycan portion of the surface lipophosphoglycan from Leishmania donovani (the disaccharide) or Leishmania major (all three compounds), were prepared and used as TLC standards to help the identification and differentiation of the elongating and branching beta-D-galactosyl transferase activities in Leishmania. The phosphosaccharides were synthesised using the H-phosphonate method for phosphorylation.     

Angiotensin II Inactivation Process in Cultured Mouse Spinal Cord Cells

The pattern of hydrolysis of [3H]angiotensin II ( [3H]AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37 degrees C. In the absence of peptidase inhibitors, 70% of [3H]AII was degraded, and the main labeled metabolite was [3H]tyrosine (40% of total radioactivity). Minor quantities of [3H]AII1-5 and [3H]AII4-8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the AII-hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4.14.4) could play a critical role, as suggested by the formation of [3H]Val3-Tyr4 and [3H]-Tyr4-Ile5 in the presence of bestatin (2 X 10(-5) M). This hypothesis was confirmed by the potency of dipeptidyl amino-peptidase III inhibitors to inhibit both [3H]AII hydrolysis and formation of these 3H-labeled dipeptides. An arylamidase-like activity could also be participating in [3H]AII hydrolysis, because higher concentrations of bestatin (10(-4) M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited [3H]tyrosine formation, increased protection of [3H]AII and [3H]AII1-7 formed, and provoked a slight accumulation of [3H]AII2-8. These results suggest that the formation of [3H]AII2-8 is due to the action of a bestatin-insensitive acidic aminopeptidase and that the Pro7-Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning, expression, and characterization of a beta-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303

The beta-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDDeltafull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDDeltafull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.     

Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product

The gene for β-agarase of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis strain DB104 for efficient and economical mass-production of the enzyme. We isolated 360 mg protein with a specific activity of 201 U/mg from the culture broth. The efficiency of production was approximately 130-fold higher than that in E. coli. The enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose from agar. Neoagarooligosaccharides produced by the enzyme had a whitening effect and inhibited tyrosinase activity in the murine melanoma cell line, B16F10. Neoagarooligosaccharides were not cytotoxic to B16F10 or normal cells. β-Agarase could therefore be a good whitening, cosmetic additive.     

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.     

Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-delta 4,5-glycuronate-terminated oligosaccharides from heparin

The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-alpha-L-threohex-4-enopyranosyl uronic acid-(1----4)-2-deoxy-2-sulphamido-6-O-sulphato-D-glucose (delta UA-2S----GlcNS-6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-alpha-L-threo-hex-4-enopyranosyl uronic acid-(1----4)-2-amino-2-deoxy-6-O-sulphato-D-[1-3H]glucitol (delta UA-2S----[1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the delta 4,5-glycuronidase for heparin delta 4,5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5-6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm-3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher delta 4,5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415].     

Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2

beta-Agarase was purified from the culture fluid of a porphyran-decomposing marine bacterium (strain AP-2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0-9.0 and at temperatures below 45 degrees C. The beta-agarase was a novel endo-type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1----3)-D-galactose] as the predominant product. The enzyme did not act on kappa-carrageenan. According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.