Characterization and substrate specificity of an endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular multienzyme complex of Bacillus circulans.

An endo-1,4-beta-D-glucanase I (Avicelase I; EC 3.2.1.4) was purified to homogeneity from an extracellular celluloxylanosome of Bacillus circulans F-2. The purification in the presence of 6 M urea yielded homogeneous enzyme. The enzyme had a monomeric structure, its relative molecular mass being 75 kDa as determined by gel filtration and 82 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.4, and the N-terminal amino acid sequence was ASNIGGWVGGNESGFEFG. The optimal pH was 4.5, and the enzyme was stable at pH 4 to 10. The enzyme has a temperature optimum of 50 degrees C, it was stable at 55 degrees C for 46 h, and it retains approximately 20% of its activity after 30 min at 80 degrees C. It showed high-level activity towards carboxymethyl cellulose (CMC) as well as p-nitrophenyl-beta-D-cellobioside, 4-methylumbelliferyl cellobioside, xylan, Avicel, filter paper, and some cello-oligosaccharides. Km values for birch xylan, CMC, and Avicel were 4.8, 7.2, and 87.0 mg/ml, respectively, while Vmax values were 256, 210, and 8.6 mumol x min-1 x mg-1, respectively. Cellotetraose was preferentially cleaved into cellobiose (G2) plus G2, and cellopentaose was cleaved into G2 plus cellotriose (G3), while cellohexaose was cleaved into cellotetraose plus G2 and to a lesser extent G3 plus G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. Xylotetraose (X4) and xylobiose (X2) were mainly produced by the enzyme hydrolysis of xylan. G2 inhibited the activity of carboxymethyl cellulase and Avicelase, whereas Mg2+ stimulated it. The enzyme was completely inactivated by Hg2+, and it was inhibited by a thiol-blocking reagent. Hydrolysis of CMC took place, with a rapid decrease in viscosity but a slow liberation of reducing sugars. On the basis of these results, it appeared that the cellulase should be regarded as endo-type cellulase, although it hydrolyzed Avicel.     

The Loss of a Sugar Chain at C(3) Position Enhances Stemucronatoside K‐Induced Apoptosis,Cell Cycle Arrest,and Hedgehog Pathway Inhibition in HT‐29 Cells

Stemucronatoside K (SMK) and its aglycone stephanthraniline A (STA) are the most representative of a series of novel C₂₁ steriodal compounds that we have previously isolated from Asclepiadaceae plants. The objectives of this study were to investigate the antitumor activity of SMK and STA, and clarify the effect of the sugar chain at the C(3) position. Our results showed that both SMK and STA decreased the growth of HT‐29 cells in a dose‐ and time‐dependent manner. Meanwhile, STA showed much stronger inhibitory effect than SMK. Treatment of HT‐29 cells with STA increased the apoptotic cell numbers and the protein expression of cleaved caspase 3 and cleaved‐PARP. G1 phase cell cycle arrest and decreased expression of cyclin D1 and cyclin‐dependent kinases 4 were also observed after STA treatment. Furthermore, STA reduced the mRNA levels of four Hedgehog pathway components (GLI1, GLI2, GLI3, and PTCH1) and suppressed Shh‐induced Hedgehog pathway activation in a concentration‐dependent manner. These results indicated that SMK and STA could inhibit the growth of HT‐29 cells by inducing apoptosis, cell cycle arrest, and hedgehog pathway inhibition. The loss of sugar chain at C(3) position could enhance SMK's activity. This study is beneficial to understand the use of natural C₂₁ steroids as antitumor lead compounds.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Endonucleolytic cleavage of E. coli and pBR 322 DNAs by a placental DNA-binding protein

A 34,000 dalton DNA-binding protein (DBP) has been purified from human placenta. The purified protein possesses endonuclease activities capable of cleaving plasmid pBR 322 and chromosomal DNAs from E. coli. Maximum endonuclease activity was observed in the pH range of 6-9 and at 30 degrees C. The nuclease activity of the DBP was completely lost at 50 degrees C. Nitrocellulose filter binding assays indicate preferential binding of the DBP to ss DNA. The protein did not bind to apurinic DNA and UV-irradiated ds DNA. Consistent with the lack of binding of the DBP to apurinic DNA, this substrate was not cleaved by the DBP. However, native and UV-irradiated E. coli DNAs which showed poor binding were also cleaved by the DBP.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site.     

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.     

Isolation and characterization of endonuclease J: a sequence-specific endonuclease cleaving immunoglobulin genes.

An endonuclease activity which cleaves close to the recombination sites of the immunoglobulin JK segments was found in extracts of chicken bursa of Fabricius and characterized after partial purification. The enzyme preparation also cleaved a VK segment at its 3' end. A similar activity was found in mouse liver, mouse myelomas and Hela cells. The enzyme designated as endonuclease J introduces double-stranded cleavages preferentially at sequences containing G clusters of pBR322 as well as the JK segments. However, not all the G clusters were cleaved by endonuclease J, suggesting that the enzyme recognizes additional sequences. Deletion of the conserved nonamer (GGTTTTTGT) located immediately 5' to the JK4 segment drastically reduced the cleavage activity of its immediate downstream G cluster. Although biological function of endonuclease J is not clear at this stage, the possibilities of its involvement in the immunoglobulin gene recombination and general recombination were discussed.     

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

The cleavage site specificity of human prostate specific antigen for insulin-like growth factor binding protein-3

The cleavage site of human insulin-like growth factor binding protein-3 by urinary prostate specific antigen was examined. Human insulin-like growth factor binding protein-3 was incubated with urinary prostate specific antigen at 37 degrees C and its proteolyzed fragments were separated by a reversed phase HPLC followed by N-terminal amino acid sequence analysis, demonstrating that the cleavage mainly occurred at Tyr-159. The synthetic peptide including Tyr-159 was also cleaved at the same site, although its reaction rate was relatively low. These results indicate that human insulin-like growth factor binding protein-3 is specifically cleaved at Tyr-159 by prostate specific antigen. Human insulin-like growth factor binding protein-3 was previously reported to be cleaved at five sites including Arg-97, Arg-132, Tyr-159, Phe-173 and Arg-179 by another group, however, prostate specific antigen preparation is possibly contaminated by trypsin-like protease. In contrast, our purified urinary prostate specific antigen had only a chymotrypsin-like activity, demonstrating that prostate specific antigen has the high substrate specificity for human insulin-like growth factor binding protein-3.     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental substantiation of the possibility to obtain anti-Proteus plasma]

The possibility to produce the anti-Proteus plasma is experimentally substantiated. It is determined that polyvalent Proteus antigen immunization permits producing blood immune preparation, that is the high-active anti-Proteus plasma. its antibodies belonging to the class of immunoglobulin G. The anti-Proteus plasma in liquid (at t = 6 degrees divided by 2 degrees C) and in frozen (at t = 20 degrees-5 degrees C) forms retains the specific activity for 3 days and 6 months, respectively.     

A site-specific endonuclease from the thermophilic strain Bacillus species F4.

A strain producing the site-specific endonuclease BspF4I was found during screening of thermophilic bacteria isolated from soil. The restriction endonuclease, free from contaminant nonspecific nucleases, was purified using three steps of column chromatography--on hydroxyapatite, blue agarose, and DEAE-Trisacryl. The enzyme is stable on storage and exhibits maximal activity at 48-56 degrees C in the presence of albumin in buffer containing 10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2. BspF4I recognizes the sequence 5;-GGNCC-3; on DNA and is an isomer and not an isoschizomer of the endonuclease Sau96I. Unlike the prototype, BspF4I does not cleave the site in a defined way. A strand with purine in the center of the sequence is cleaved after the first G, as in the case of the prototype, while the strand with pyrimidine is cleaved either before or after the first G.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Molecular cloning of the Lon protease gene from Thermus thermophilus HB8 and characterization of its gene product.

The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is approximately 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of approximately 5 mg.L-1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and alpha-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 degrees C, which was significantly higher than the optimal temperature (37 degrees C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.