Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.     

Transport of ascorbate into guinea pig liver mitochondria

The amount of ascorbate associated with guinea pig liver mitochondria was estimated by high-performance liquid chromatography. Incubation of mitochondria with ascorbate revealed a time-dependent and temperature-dependent accumulation of the vitamin. A steady-state level of ascorbate was obtained in the mitochondria after about 20 min of incubation at 37 degrees C, whereas no accumulation was observed at 0 degrees C. The matrix concentration of ascorbate was highly correlated to the concentration of ascorbate in the incubation medium. The initial rate of accumulation (about 7 pmol/mg protein per min at 10 degrees C) was three orders of magnitude less than for compounds that are transported across the mitochondrial inner membrane by specific carriers. Experiments with the enzyme ascorbate oxidase demonstrated that the mitochondrial membrane is also permeable to dehydroascorbate, and that the accumulated dehydroascorbate is stable in the mitochondria. There was no effect of the energy state of the mitochondrial membrane of the initial transport rate of ascorbate. Electrostatic binding of ascorbate to the membrane was excluded from experiments performed in isosmotic potassium chloride medium. Diffusion of ascorbate across the mitochondrial inner membrane accounts for the experimental findings.     

Sugar-binding sites with specificity for glucosamine in the guinea pig middle ear mucosa

Glucosamine-binding sites were detected in Lowicryl K4M-embedded guinea pig middle ear mucosa by electron microscopy, using glucosaminyl bovine serum albumin. Incubation of ultrathin tissue sections with gold-labeled glucosaminyl bovine serum albumin (GlcN/BSA/gold) resulted in binding mainly on cilia, microvilli, rough endoplasmic reticulum and nuclei. The sugar binding was not inhibited after ultrathin sections had been digested with trypsin or neuraminidase. Various carbohydrates and glycoconjugates were tested as competitive inhibitors of GlcN/BSA/gold labeling on the tissue sections. The sugar specificity range detected by the glucosamine-binding sites included glucosamine, N-acetylglucosamine, mannose and fucose, whereas N-acetylgalactosamine, galactose and glucose were not detectable. A series of endotoxic substances such as Salmonella minnesota Re595 lipid A complex with BSA and lipopolysaccharides (LPS) derived from Escherichia coli 055:B5 or S. minnesota Re595 also competed with GlcN/BSA/gold binding. This indicates that the lipid A backbone glucosamine or other carbohydrate portions of LPS is a part of the structure recognized by glucosamine-binding sites.     

Nonparticipation of adrenergic mechanism in potassium-induced relaxation of taenia from guinea pig caecum

In the isolated taenia caeci of guinea pigs excess potassium (10-30 mM) induced a phasic relaxation followed by a contraction. This phasic relaxation was unaffected by treatment with hexamethonium, phentolamine, propranolol and bretylium. However, relaxation induced by perivascular nerve stimulation was inhibited by all these agents but not by hexamethonium. Tetrodotoxin inhibited both forms of relaxation. Potassium-induced relaxation was not accompanied by [3H]noradrenaline release. Perivascular nerve stimulation caused release of [3H]noradrenaline and this was blocked by bretylium. Treatment with ouabain or replacement of NaCl by LiCl, but not treatment by cold storage, inhibited the potassium-induced relaxation. These results suggest that the potassium-induced relaxation of taenia caeci was due to electrogenic sodium pumping and was independent of the adrenergic innervation of the tissue.     

Purification and characterization of a microsomal bile acid beta-glucosidase from human liver

A human liver microsomal beta-glucosidase has been purified to apparent homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis where a single protein band of Mr 100,000 was obtained under reducing conditions. The enzyme was enriched about 73, 000-fold over starting microsomal membranes by polyethylene glycol fractionation, anion exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by affinity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhibition of activity. The enzyme catalyzed the hydrolysis of 3beta-D-glucosido-lithocholic and 3beta-D-glucosido-chenodeoxycholic acids with high affinity (Km, 1.7 and 6.2 microM, respectively) and of the beta-D-glucoside (Km, 210 microM) and the beta-D-galactoside of 4-methylumbelliferone. The ratio of relative reaction rates for these substrates was about 6:3:11:1. No activity was detectable toward 6beta-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta-D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitation studies using antibodies prepared against lysosomal glucocerebrosidase showed no cross-reactivity with microsomal beta-glucosidase suggesting that these two enzymes are antigenically unrelated.     

Large-scale preparation of cytosolic and mitochondrial asparatate aminotransferases from pig heart

A procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heat muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2 g of the cytosolic isoenzyme and 1.7 g of the mitochondrial isoenzyme were obtained from 20 kg of minced heart muscle.     

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.     

The mechanism and site of action of lidocaine hydrochloride in guinea pig inner ear

Lidocaine was applied to the round window (RW) in order to localize its site of action in the cochlea. Cochlear microphonic (CM), summating potential (SP), and compound action potential (CAP) input/output functions were measured to a 16 kHz tone burst to assess the functional changes of the cochlea. In separate experiments, the effect of lidocaine on the whole cell current of isolated outer hair cells (OHC) was studied. A dose of 2 microliters of 40 mM lidocaine in saline solution, when applied to the RW, caused a small change in all measured variables, indicating a passage of the drug through the RW membrane to sites of action. However, 160 mM of lidocaine further decreased CM, SP, and CAP by a total of 40% from the control. A partial recovery occurred for CM during the 30 min follow-up period. CAP and SP continued to decline. In isolated OHCs, lidocaine decreased the whole cell current in a dose-dependent fashion. The KD for lidocaine effect on OHCs was 7 mM. Our in vivo results indicate that lidocaine affects OHCs and reduces CM, causing a subsequent reduction in SP and CAP. The increased effect of lidocaine on CAP and SP, while CM is recovering, suggests an additional specific effect of lidocaine on the cochlear nerve and/or on inner hair cells. Considering that lidocaine alters OHC current (in isolated hair cells) and that lidocaine does not affect endocochlear potential [Laurikainen et al. Acta Otolaryngol (Stockh) 1991: 112: 800-9], the observed CM changes are most likely due to an in vivo effect on OHCs. Thus, the early effect of lidocaine on the cochlea appears to be due to a significant change in organ of Corti function, rather than to direct anesthesia of the cochlear nerve. Later, an independent effect of the drug may occur on neural tissues in the inner ear.     

Anti-atrial fibrillation effects of cyclovirobuxine-D and its electrophysiological mechanism studied on guinea pig atria

Cyclovirobuxine-D (CVB-D) was shown to produce significant and dose-dependent protective effects against atrial fibrillation induced by CaCl2-Ach in mice. On atrial fibrillation induced by aconitine, ouabain or adrenaline in isolated guinea pig atria, the effects of CVB-D were similar to those of amiodarone. CVB-D 0.3-100 mumol.L-1 was shown to depress the automaticity of the isolated guinea pig right atria. In isolated left atria, CVB-D 0.3 mumol.L-1 was found to inhibit the abnormal automaticity elicited by adrenaline, to prolong the duration of action potential and effective refractory period and to reduce excitability. At high concentration (30 mumol.L-1), CVB-D was also found to decrease the maximal velocity of depolarization (Vmax) and to elongate the conduction time of initiation. Amiodarone 0.3-30 mumol.L-1 was shown to closely resemble CVB-D in electrophysiology without effect on Vmax.     

Isolation of an atypical Pasteurella-like organism from guinea pig abscesses

The action of eserine of acetylcholinesterase (AchE) reversible inhibitor of frog muscle fibres membrane potential (MP) under various physico-chemical conditions in external solution was studied. The data obtained show that changes of external pH in any direction decrease the depolarisation of the membrane produced by eserine. The dose-effect curve is linear at pH 7, but it has saturation at pH 6 and pH 9. Dependence of the membrane depolarisation in the presence of eserine upon calcium ions concentration in external solution is S-shape. Protonophore 3C1CCP (carbonilcianamid-m-3C1-phenylhydrazon) depolarises the membrane further in the presence of eserine. Valinomycin under these conditions completely restores the MP. Evidence is obtained that eserine reduces potassium permeability of the muscle membrane. It is supposed that membrane-bound AchE is involved in the ionic permeability regulation of the muscle membrane at rest.     

Regulatory mechanism of pepsinogen gene expression in monolayer cultured guinea pig gastric chief cells

We have investigated the effects of dibutyryl cAMP, forskolin, carbamylcholine chloride (carbachol), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of guinea pig pepsinogen mRNA in monolayer cultured gastric chief cells. After exposure of the cells to each of these compounds for 4 to 24 hr, and at 48 hr after primary culture, total cellular RNA was isolated using acid guanidium-phenol-chloroform and then was reverse transcribed to cDNA. Obtained cDNA was amplified by polymerase chain reaction (PCR) using primers detecting guinea pig pepsinogen mRNA and human beta-actin mRNA as an internal standard. The PCR products were separated and quantified using capillary electrophoresis. Dibutyryl cAMP and forskolin significantly increased pepsinogen mRNA, but carbachol, ionomycin, and TPA failed to increase that. These findings suggested that pepsinogen gene expression was up-regulated by intracellular cAMP, but not by intracellular calcium or protein kinase C in guinea pig chief cells.     

Circularization and cleavage of guinea pig cytomegalovirus genomes

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.     

ATP release evoked by isoprenaline from adrenergic nerves of guinea pig atrium

Mode and site of release of ATP evoked by isoprenaline were evaluated in the electrically driven left atrial segment of guinea pig. The peak release of ATP 5 min after 1 microM isoprenaline was inhibited by 1 microM propranolol and 1 microM butoxamine, but not by 1 microM atenolol, showing that the ATP release is due to stimulation of the presynaptic beta 2-adrenoceptor by isoprenaline. The maximum ATP release was markedly reduced by Ca2+/calmodulin antagonists, W-7 and trifluoperazine, and by a mitotic inhibitor, vinblastine. Further, the release was similarly inhibited by myosin light chain kinase inhibitors, ML-7 and wortmannin. Nifedipine, a Ca(2+)-channel blocker, decreased the release of ATP evoked by isoprenaline. By contrast, Bay K 8644, a Ca(2+)-channel opener, tended to enhance the ATP release. These findings suggest that isoprenaline produces ATP release from adrenergic nerve terminals of atrium, implying that ATP serves as a co-transmitter.     

Effects of raloxifene in a guinea pig model for leiomyomas

OBJECTIVE: Chronic exposure of oophorectomized guinea pigs to 17beta-estradiol causes leiomyoma formation. Our aims were to determine whether these leiomyomas can become estradiol independent after exposure to estradiol and if raloxifene inhibits leiomyoma growth when given concomitantly with estradiol. STUDY DESIGN: To induce leiomyoma development, 6 oophorectomized animals received two estradiol implants for 140 days. Next, the estradiol implants were replaced with empty implants in 3 animals, whereas the other 3 received 2 new estradiol implants and raloxifene given per os 10 mg/kg per day for 60 days. Tumor size was monitored biweekly by ultrasonography. RESULTS: On estradiol removal, abdominal wall leiomyomas regressed within 15 to 30 days; when estradiol implants were reintroduced, leiomyomas redeveloped. Within 30 days on raloxifene, all abdominal leiomyomas (n = 9) regressed as determined by ultrasonography and verified at laparotomy. Serum raloxifene and estradiol levels were 432 +/- 46 pg/mL and 78 +/- 13 pg/mL (mean +/- SEM, n = 3), respectively, after 60 days of treatment. CONCLUSIONS: Leiomyomas did not become estradiol independent, even after long exposure to estradiol; ultrasonography allowed frequent, noninvasive assessment of leiomyoma size, and raloxifene rapidly regressed leiomyomas in this animal model.