Isolation of achatin-I, a neuroactive tetrapeptide having a D-phenylalanine residue, from Achatina ganglia, and its effects on Achatina giant neurones.

1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly-D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani et al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50 micrograms of achatin-I and 17 micrograms of its stereoisomer consisting of only L-amino acid residues (Gly-L-Phe-L-Ala-L-Asp) which was termed achatin-II. The data of instrumental analyses (1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, [D-Ala3]-achatin-I (Gly-D-Phe-D-Ala-L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [D-Ala3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage (Vh) of -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Férussac containing a D-amino acid residue

A tetrapeptide named achatin-I was purified from the suboesophageal and cerebral ganglia of the African giant snail Achatina fulica Férussac, and evoked a potent neuroexcitatory effect. The amino acid sequence of achatin-I is Gly-D-Phe-Ala-Asp. Achatin-I induced a voltage-dependent inward current, due to Na+, on the identifiable giant neuron, periodically oscillating neuron (PON), of the same snail. All possible isomers of achatin-I were synthesized using the solid-phase method. The sensitivity of the neuron to achatin-I and its isomers was strictly stereospecific; among the various isomers, only achatin-I showed marked effects (ED50 = 2.29 x 10(-6)M), while Gly-D-Phe-D-Ala-Asp, the synthetic D-Ala-isomer, was less than 10(-3) active.     

Crystal structure and molecular conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide containing a D-amino acid residue.

In order to investigate the active conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide from the Achatina fulica ganglia, its crystal structure and molecular conformation were analysed by the X-ray diffraction method. Crystals from methanol/dioxane are monoclinic, space group P2(1) with a = 5.083(1), b = 9.125(1), c = 20.939(3) A, beta = 94.73(1) degrees. The structure was solved by direct methods and refined to R = 0.051 for 1714 independent reflections with /Fo/ greater than sigma (Fo). The molecule exists as a zwitterion with the Gly N-terminal end protonated and Asp beta-carboxyl deprotonated; the C-terminal of Asp is in a neutral state. The molecule takes a kind of beta turn structure with the D-Phe-Ala residues at the corner of the bend. This turn conformation is primarily formed by the strong intramolecular hydrogen bonds of NH(Gly)...O delta 1 (Asp) and NH(Asp)...O delta 1 (Asp) pairs, thus forming a 15-membered ring structure. Judging from the published data concerning the structure-activity relationship, this turn conformation may reflect an important feature related to the neuroexcitatory activity of achatin-I.     

Structure-activity relationships of hydrazono derivatives of biological interest

The following hydrazono derivatives (I-XXIII) of type (A), (formula; see text) where: X = NO2 (II, IV, VI, VIII, X, XIV-XXIII), X = H (I, III, V, VII, IX, XI, XII, XIII), and Y = H (I, II); 3-Cl (III, IV); 4-Cl (V, VI); 3,4-Cl2 (VII, VIII); 2,6-Cl2 (IX, X); 2-NO2 (XI); 3-NO2 (XII); 4-NO2 (XIII, XIV); 2-F (XV); 3-F (XVI); 4-F (XVII); 2-OH (XVIII); 4-OH (XIX); 2,4-(OH)2(XX); 2,4,6-(OH)3(XXI); 2,3-(OH,NO2) (XXII); 2,4-(NO2)2 (XXIII), were prepared and tested for antibacterial and antifungal activity. All of these compounds were prepared in satisfactory yield by reaction of aromatic aldehydes with 2-furoyl and 5-nitro-2-furoyl hydrazide. The hydrazono derivatives I-XXIII prepared in this investigation were screened for antimicrobial activity by a disk-diffusion assay (Kirby-Bauer modified). The organisms used were laboratory cultures of S. aureus, S. -haemoliticus, B. subtilis, M. paratuberculosis, E. coli, S. typhi, Ps. aeruginosa, K1. pneumoniae, A. niger, S. cerevisiae, C. albicans. The results of this study showed that a number of the prepared hydrazono derivatives exhibited varying degrees of activity against Gram-positive and Gram-negative bacteria. Compounds IV and XV possessed broad spectrum "in vitro" against Gram-positive and Gram-negative bacteria. Compounds XII greater than IV greater than XV showed inhibitory activity especially toward S. aureus. Compounds IV greater than XV greater than XVI were especially active against E. coli. Compounds XV greater than IV were especially inhibitory toward S. typhi and most of the prepared compounds inhibited considerably Ps. aeruginosa and K1. pneumoniae.     

Anticholinesterase activity of potential therapeutic 5-(1,3,3-trimethylindolinyl) carbamates.

Six N-alkyl and N-aryl 5-(1,3,3-trimethylindolinyl) carbamates were synthesized and studied for their structure-activity relationships in inhibiting eel acetylcholinesterase (AChE). The carbamates were 5-(1,3,3-trimethylindolinyl)N,N-dimethylcarbamate (Cui Xing Ning) (I), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-ethylcarbamate (III), 5-(1,3,3-trimethylindolinyl)N,N-diethylcarbamate (IV), 5-(1,3,3-trimethylindolinyl)N-heptylcarbamate (V), and 5-(1,3,3-trimethylindolinyl)N-(3-chlorophenyl)carbamate (VI). The inhibition studies were carried out at 25.0 degrees C at pH 7.60. The rank order of the ki values for eel AChE inhibition is II > V > I > III > VI > IV. Compound II has a greater affinity for the enzyme than any irreversible inhibitor cited in the literature (Kd = 7.14 x 10(-8) M). Our findings should aid in the application of these carbamates (1) for counteracting the cholinergic problems associated with various diseases, and (2) for developing potential pretreatment compounds for organophosphate poisoning.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 183–197, 1998     

Studies on heterocyclic compounds: 1,3-thiazolidin-4-one derivatives. IV. Biological activity of variously substituted 2,3-diaryl-1,3-thiazolidin-4-ones

The following 2,3-diaryl-1,3-thiazolidin-4-ones of general formula (A) were synthesized and screened for antimicrobial activity. (formula; see text) where: X = H (I, III, V, VII, IX, XI, XIII, XV, XVII, XIX, XXI, XXIII), CH3 (II, IV, VI, VIII, X, XII, XIV, XVI, XVIII, XX, XXII, XXIV); R = H (I, II, V, VI, VII, VIII, XI, XIII), 4-CH3 (XXI, XXII, XXIII, XXIV), 4-Br (III, IV, IX, X), 2-NO2 (XIII, XIV), 3-NO2 (XV, XVI), 4-NO2 (XVII, XVIII), 4-OCH3 (XIX, XX); R' = H (I, II, III, IV, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII), 4-CH3 (XXIII, XXIV), 3-Br (V, VI), 4-Br (VII, VIII, IX, X), 4-J (XI, XII). These compounds were prepared by the general synthetic procedure previously reported for the 1,3-thiazolidin-4-one derivatives already prepared and screened in this SARs program. The synthetic approach involves the cyclocondensation of the appropriate Schiff bases with alpha-mercaptoalkanoic acids. The prepared compounds were screened against S. aureus, S. beta-haemolititicus, B. subtilis, M. paratuberculosis 607, S. typhi, Kl. pneumoniae, E. coli Bb, Ps, aeruginosa, C. albicans, A. niger, S. cerevisiae by a disk-diffusion assay (Kirby-Bauer modified). The results obtained in this investigation showed that the prepared compounds exhibited varying degrees of antimicrobial activity. They were especially inhibitory toward Gram-positive bacteria, and fungi. 4-Nitroderivatives (XVII), (XVIII), and 2-nitroderivatives (XIV) and (XIII) possessed marked antimicrobial activity against S. aureus, S. beta-haemoliticus, and B. subtilis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on heterocyclic compounds: spiro [indole-3,2'-thiazolidine] derivatives. Antimicrobial activity of monohalogenated 3'-phenylspiro 3H-indole-3,2'-thiazolidine-2,4' (1H)-diones

The following halogenated 3'-phenyl [3H-indole-3,2'-thiazolidine]-2,4'(1H)-dione of general formula (A) were synthesized and screened for antimicrobial activity. (formula: see text) where: X = H (I, III, V, VII, IX, XI, XIII, XV), CH3 (II, IV, VI, VIII, X, XII, XIV, XVI); Y = H (I, II), 3-F (III, IV), 2-Cl (V, VI), 3-Cl (VII, VIII), 4-Cl (IX, X), 2-Br (XI, XII), 3-Br (XIII, XIV), 4-Br (XV, XVI). The synthetic approach involves the preparation of variously substituted Schiff-bases of indol-2,3-dione, which then are subjected to cyclocondensation with alpha-mercaptoalkanoic acids, to give spirothiazolidinones of type (A). The prepared compounds were screened against S. aureus, B. cereus, M. paratuberculosis, E. coli, S. typhi, Pr. mirabilis, Ps. aeruginosa, C. albicans, S. cerevisiae, A. niger by a disk-diffusion assay (Kirby-Bauer modified. The results of the antimicrobial screening showed that the prepared compounds exhibited varying degrees of activity against Gram-positive, Gram-negative bacteria, and fungi. 3-Fluoro-derivative (III) showed inhibitory activity especially toward S. aureus and C. albicans. Chloroderivatives (VII) and (VIII) showed broad-spectrum "in vitro" antimicrobial activity, and were especially inhibitory toward S. aureus, E. coli, and S. Typhi. Fluoro-derivative (IV) and bromo-derivatives (XIII) and (XIV) possessed marked antimicrobial activity against M. paratuberculosis.     

Characterization of opioid receptors on isolated canine gallbladder smooth muscle cells

Smooth muscle cells were isolated from the fundus of the canine gallbladder and examined for the presence of opioid receptors. The cells contracted in a concentration-dependent manner in response to three opioid peptides (Met-enkephalin, dynorphin1-13 and Leu-enkephalin), which are known derivatives of opioid precursors present in myenteric neurons of the gut. The order of potency was Met-enkephalin greater than dynorphin1-13 greater than Leu-enkephalin. The contractile response to opioid agonists was selectively inhibited by opioid antagonists (naloxone and Mr2266) but not by muscarinic, CCK/gastrin or tachykinin antagonists. Equivalent responses to the three opioid peptides exhibited differential sensitivity to preferential antagonists of mu (naloxone) and kappa (Mr2266) opioid receptors consistent with the presence of the three main types of opioid receptors (mu, delta and kappa) on canine gallbladder muscle cells.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).     

Changes in plasma estradiol, progesterone and LH level in the menstrual cycle of the adult female Japanese monkey during the mating season

Adult 15 female Japanese monkeys showing regular menstrual cycles were subjected to the daily blood sampling for the measurement of estradiol (E2), progesterone (P) and biological LH in the mating season. Monkeys were maintained under controlled conditions in a standardized environment. Of the 35 cycles observed, 18 (51.4%) were estimated as anovulatory cycles and 17 (48.6%) were ovulatory cycles. The anovulatory cycles were classified into three types according to the peak level of E2 (Type I: E2 less than 50 pg/ml 3 cycles, Type II: E2 less than 170 pg/ml 7 cycles, Type III: E2 greater than 170 pg/ml 8 cycles). The ovulatory cycles were classified into two Types according to the peak level of P (Type IV: P less than 5.0 ng/ml 5 cycles, Tyep V: P greater than 5.0 ng/ml 12 cycles). The menstrual cycle was 27.5 +/- 7.8 days. The differences between mid cycle LH surge and P level in Type IV and in Type V were statistically significant. It was revealed that female Japanese monkeys kept under controlled condition in the mating season showed high incidence of various types of anovulatory cycles and that the ovulatory cycles with low P elevation in the mid luteal phase showed low LH and P secretions on the mid cycle date.     

Effects of synthetic peptides on giant neurons identified in the ganglia of an African giant snail (Achatina fulica Férussac)--II

Thirteen synthetic biologically-active peptides, which were classified into the peptides proposed as neurotransmitters in mammals and invertebrates and neural venom peptides, were investigated for their effects on the following six identifiable giant neurons of an African giant snail (Achatina fulica Férussac): RAPN (right anterior pallial neuron), INN (intestinal nerve neuron), RPeNLN (right pedal nerve large neuron), LPeNLN (left pedal nerve large neuron), d-LPeLN (dorsal-left pedal large neuron) and d-LPeCN (dorsal-left pedal constantly firing neuron). Oxytocin and proctolin at 10(-4)M excited the RAPN membrane potential, whereas FMRFamide at the same concentration inhibited the same neuron. FMRFamide at 10(-4)M markedly inhibited the d-LPeLN membrane potential, sometimes produced inhibition of RPeNLN and LPeNLN, showed varied effects (excitatory or inhibitory) on INN, and had no effect on d-LPeCN. The other peptides examined had almost no effect on any of the neurons tested.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

Interaction of elongation factor EF-Tu with gamma-amides of GTP and beta-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: gamma-(4-azido)anilide of GTP (I), gamma-(n-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: beta-(4-azido)anilide of GDP (III), beta-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N-beta-(4-methylmorpholinium)-ethylcarbodiimide p-toluene sulfonate and the respective amine. The analog of GTP bearing at the gamma-phosphate an alkylating 2-chloroethylamino group: gamma-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105-108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu.GDP with the respective Ki values 3.9.10(-7) M (I), 2.9.10(-8)M (II), 6.9.10(-7)M (III), 5.0.10(-7)M (IV) and 3.8.10(-8)M (V) relative to GDP. The dissociation constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5.10(-6)M, 3.4.10(-7)M and 4.6.10(-8)M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.     

Collagen XVI induces formation of focal contacts on intestinal myofibroblasts isolated from the normal and inflamed intestinal tract

In Crohn's disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell–matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin α1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of α1β1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell–matrix interaction preferentially through recruitment of α1ß1 integrin into the tips of the focal adhesion contacts.     

Ulnar polydactyly

A retrospective review of 148 patients with ulnar polydactyly was conducted to analyze the types, patterns of involvement, associated anomalies, treatments, and outcomes of this malformation. The hands only were involved in 123 patients, both hands and feet in 20 patients, and five patients had mixed radial and ulnar polydactyly. Ulnar polydactyly was more prevalent among males. Among African Americans, the condition was often bilateral. When unilateral, ulnar polydactyly occurred more often on the left side. The racial distribution was 103 African Americans (70 percent), 37 Caucasians (25 percent), four Native Americans, three Latin Americans, and one Asian. Five types were encountered: type I cutaneous nubbin, type II pedunculated digit, type III articulating digit with fifth metacarpal, type IV fully developed digit with sixth metacarpal, and type V polysyndactyly. The distribution of types in order of frequency was type II, III, V, I, and IV. Types I and II ulnar polydactyly combined were more prevalent (82 percent) than types III, IV, and V (18 percent). Types I and II were more common among African Americans. Types III, IV, and V ulnar polydactyly occurred more frequently among Caucasians, but these were slightly less prevalent than types I and II in this racial group. Five patients were syndromic; four were Caucasians, and one Asian. Most cases of ulnar polydactyly of the hand were treated by ligation (71 percent) in the nursery, whereas polydactyly of the foot was more often referred to a specialist to be treated by surgical ablation (92 percent). Treatment complications occurred more frequently in the hands than in the feet. The complication rate after ligation of ulnar polydactyly of the hand was 23.5 percent. The two main complications were tender or unacceptable nubbins and infections.     

Synthesis of guanosine and its derivatives from 5-amino-1-beta-D-ribofuranosyl-e-imidazolecarboxamide. IV. A new route to guanosine via cyanamide derivative.

4-Cyanamido-5-imidazolecarboxamide (IV) was prepared by brief treatment of 5-(S-methylisothiocarbamoyl) amino-4-imidazolecarboxamide (V) with alkali. Compound VI was converted in an alkaline solution to either guanine (VII) or isoguanine (VIII), depending on the concentration of alkali. This procedure was applied to the synthesis of 2',3'-0-isopropylideneguanosine (XVI) from the riboside of 5-(N'-benzoyl-S-methylthiocarbamoyl) amino-4-imidazolecarboxamide (IX), PROviding a new route to XVI.     

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.