Journal Article Enhancement of Activities of Cellulases under High Hydrostatic Pressure Get access Sawao Murao, Sawao Murao Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Yoshiyuki Nomura, Yoshiyuki Nomura Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Miki Yoshikawa, Miki Yoshikawa Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Takashi Shin, Takashi Shin Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Hiroshi Oyama, Hiroshi Oyama Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Motoo Arai Motoo Arai Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, JapanDepartment of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai 591, Japan Search for other works by this author on: Oxford Academic Google Scholar Bioscience, Biotechnology, and Biochemistry, Volume 56, Issue 8, 1 January 1992, Pages 1366–1367, https://doi.org/10.1271/bbb.56.1366 Published: 01 January 1992 Article history Published: 01 January 1992 Received: 23 March 1992
Restoration of cranial nerve functions during acoustic neuroma (AN) surgery is crucial for good outcome. The effects of minimizing the injury period and maximizing the recuperation period were investigated in 89 patients who consecutively underwent retrosigmoid unilateral AN surgery.Cochlear nerve and facial nerve functions were evaluated during AN surgery by use of continuous auditory evoked dorsal cochlear nucleus action potential monitoring and facial nerve root exit zone-elicited compound muscle action potential monitoring, respectively. Factors affecting preservation of function at the same (preoperative) grade were analyzed.A total of 23 patients underwent standard treatment and investigation of the monitoring threshold for preservation of function; another 66 patients underwent extended recuperation treatment and assessment of its effect on recovery of nerve function. Both types of final action potential monitoring response and extended recuperation treatment were associated with preservation of function at the same grade.Preservation of function was significantly better for patients who received extended recuperation treatment.
We previously developed a system with which we have created more than 100 virtual cancer images from CT or MR data of individual patients with cancer (Cancer Edutainment Virtual Reality Theater: CEVRT). These images can be used to help explain procedures, findings, etc. to the patient, to obtain informed consent, to simulate surgery, and to estimate cancer invasion to surrounding organs. We recently developed a web-based object-oriented database both to access these cancer images and to register medical images at international research sites via the Internet. In this report, we introduce an international medical VR data warehouse created using an object-oriented database.
Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser278-Glu78-Asp82) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1′, showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser278, His78, and Asp82 do not interact with each other; thus, the canonical catalytic triad is disrupted. Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser278-Glu78-Asp82) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1′, showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser278, His78, and Asp82 do not interact with each other; thus, the canonical catalytic triad is disrupted. A novel peptidase, initially named ScpA (1Tsuruoka N. Isono Y. Shida O. Hemmi H. Nakayama T. Nishino T. Int. J. Syst. Evol. Microbiol. 2003; 53: 1081-1084Crossref PubMed Scopus (58) Google Scholar) and now called kumamolisin-As (2Wlodawer A. Li M. Gustchina A. Oyama H. Dunn B.M. Oda K. Acta Biochim. Polon. 2003; 50: 81-102Crossref PubMed Scopus (80) Google Scholar), was recently identified by us in the culture filtrate of a thermoacidophilic soil bacterium Alicyclobacillus sendaiensis strain NTAP-1 (1Tsuruoka N. Isono Y. Shida O. Hemmi H. Nakayama T. Nishino T. Int. J. Syst. Evol. Microbiol. 2003; 53: 1081-1084Crossref PubMed Scopus (58) Google Scholar). Specificity analyses using macromolecular substrates including globular and other fibrillar proteins showed that kumamolisin-As is highly specific for collagen (3Nakayama T. Tsuruoka N. Akai M. Nishino T. J. Biosci. Bioeng. 2000; 89: 612-614Crossref PubMed Scopus (27) Google Scholar, 4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar) and thus could be considered as a collagenase, although with some unusual properties. Most noticeably, this enzyme exhibits the maximum activity at acidic pH ∼4.0. This is in striking contrast to all known collagenases, which are either zinc-dependent metallopeptidases (5Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar) or chymotrypsin-like serine proteinases (6Okamoto M. Yonejima Y. Tsujimoto Y. Suzuki Y. Watanabe K. Appl. Microbiol. Biotechnol. 2001; 57: 103-108Crossref PubMed Scopus (74) Google Scholar, 7Tsu C.A. Perona J.J. Fletterick R.J. Craik C.S. Biochemistry. 1997; 36: 5393-5401Crossref PubMed Scopus (38) Google Scholar), with an optimum pH for activity at neutral to alkaline regions. A primary structure analysis of this novel "acid collagenase" revealed that it is a member of the sedolisin family, a recently established class of serine peptidases with a unique catalytic triad, Ser-Glu-Asp, in place of the Ser-His-Asp triad of classical serine peptidases (2Wlodawer A. Li M. Gustchina A. Oyama H. Dunn B.M. Oda K. Acta Biochim. Polon. 2003; 50: 81-102Crossref PubMed Scopus (80) Google Scholar, 4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar). Moreover, the enzyme was found to be very similar in its primary structure to kumamolisin, a well characterized member of the family (8Murao S. Ohkuni K. Nagao M. Hirayama K. Fukuhara K. Oda K. Oyama H. Shin T. J. Biol. Chem. 1993; 268: 349-355Abstract Full Text PDF PubMed Google Scholar, 9Oyama H. Hamada T. Ogasawara S. Uchida K. Murao S. Beyer B.B. Dunn B.M. Oda K. J. Biochem. (Tokyo). 2002; 131: 757-765Crossref PubMed Scopus (22) Google Scholar, 10Comellas-Bigler M. Fuentes-Prior P. Maskos K. Huber R. Oyama H. Uchida K. Dunn B.M. Oda K. Bode W. Structure. 2002; 10: 865-876Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), exhibiting 92.7% identity with its mature form. This high level of identity led to the change of the name from the initially used ScpA (1Tsuruoka N. Isono Y. Shida O. Hemmi H. Nakayama T. Nishino T. Int. J. Syst. Evol. Microbiol. 2003; 53: 1081-1084Crossref PubMed Scopus (58) Google Scholar) to kumamolisin-As (2Wlodawer A. Li M. Gustchina A. Oyama H. Dunn B.M. Oda K. Acta Biochim. Polon. 2003; 50: 81-102Crossref PubMed Scopus (80) Google Scholar). Kumamolisin-As was the first member of the sedolisin family to be shown capable of degrading collagen, but further analysis of the substrate preferences of kumamolisin detected some collagenolytic properties, although not as pronounced (4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar). Collagen is an insoluble structural protein that accounts for ∼30% of the total weight of animal proteins (5Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar). It is the predominant constituent of skin, tendons, and cartilage as well as the organic component of bones, teeth, and the cornea. Collagen is also found in the connective tissues of nearly all organs as insoluble fibers embedded in the extracellular matrix, where it serves to provide both their structure and strength (5Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar). Enzymatic degradation of collagen has attracted medical attention because it is closely related to the etiology of many human diseases (5Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar). Considering the abundance of collagen in nature, microbial degradation of collagen should also be of biogeochemical significance in global cycling of nitrogen (11Madigan M.T. Martinko J.M. Parker J. Biology of Microorganisms. Prentice-Hall, Saddle River, NJ2000Google Scholar). Due to its rigidified fibrillar structure, collagen is not generally degraded by ordinary peptidases but can only efficiently be degraded by the collagen-specific enzymes named collagenases (5Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar). Kumamolisin-As was the first example of a collagenase from the sedolisin family, and analyses of its subsite specificity, its mode of collagen binding, and the role of its unique catalytic triad could be very interesting issues to be clarified in comparison with the classical types of collagenases. We report here the results of such a study, conducted using crystallographic and biochemical approaches. In addition, we created and characterized the E78H mutant of the enzyme, in which the glutamate residue of its catalytic triad was replaced by a histidine in order to mimic the catalytic triad of the classical serine peptidases. Collagen (type I, from bovine Achilles tendon) and high performance liquid chromatography (HPLC) 1The abbreviations used are: HPLC, high performance liquid chromatography; IQF, internally quenched fluorogenic substrate; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; NMA, N-methylanthranilic acid; DNP, 2,4-dinitrophenol; AcIPF, N-acetylisoleucyl-prolyl-phenylalaninal. grade acetonitrile were purchased from Nacalai Tesque (Kyoto, Japan). All chemicals for peptide synthesis were obtained from PerkinElmer Life Science. An internally quenched fluorogenic substrate (IQF), NMA-MGPH*FFPK(DNP)dRdR ([2-(N-methylamino)benzoyl]-l-methionyl-glycyl-l-prolyl-l-histidyl-l-phenylalanyl-l-phenylalanyl-l-prolyl-Nϵ-(2,4-dinitrophenyl)-l-lysyl-d-arginyl-d-arginine amide) was a product of the Peptide Institute (Osaka, Japan). An inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) was synthesized as described previously (9Oyama H. Hamada T. Ogasawara S. Uchida K. Murao S. Beyer B.B. Dunn B.M. Oda K. J. Biochem. (Tokyo). 2002; 131: 757-765Crossref PubMed Scopus (22) Google Scholar, 12Wlodawer A. Li M. Gustchina A. Dauter Z. Uchida K. Oyama H. Goldfarb N.E. Dunn B.M. Oda K. Biochemistry. 2001; 40: 15602-15611Crossref PubMed Scopus (50) Google Scholar). Restriction enzymes and other DNA-modifying enzymes were purchased from TaKaRa Shuzo (Kyoto, Japan) or from Toyobo (Osaka, Japan). The plasmid pScpA, which is a derivative of pET15b (Novagen, Madison, WI), was constructed as described previously (4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar) and was used for the expression of the full-length kumamolisin-As gene. All other chemicals used were of analytical or sequencing grade, as appropriate. Two methods of assessing collagenase activity were employed in this study. Method I—This assay system contained 100 μm sodium acetate, pH 4.0, 2 mg of collagen, and enzyme in a final volume of 0.5 ml. After preincubation at 60 °C, the reaction was started by the addition of the enzyme. Incubation was carried out at 60 °C with shaking (at 1000 rpm) for 10-60 min (depending on the amount of enzyme to be assayed) using a micromixer model E-36 (TAITEC Co., Saitama, Japan) that maintained the homogeneous distribution of the collagen powder in the reaction mixture during incubation. The blank reference mixture did not contain the enzyme. The reaction was stopped by adding 1.0 μl of 0.2 m HCl and chilling the mixture on ice for 15 min followed by centrifugation. Supernatant (100 μl) was mixed with 400 μl of Ninhydrin Color Reagent Solution (Nacalai Tesque) and was heated at 97 °C for 10 min, followed by chilling the mixture on ice. 2-Propanol (1.0 ml) was then added to the mixture, and the increase in absorbance of the supernatant at 570 nm (ΔA570) was determined. The ΔA570 of the reaction mixture, in which collagen (2 mg) was completely degraded by the addition of an excess amount of the collagenase under these assay conditions, was also determined and used for unit calculations. Method II—For assaying the enzymatic hydrolysis of the IQF substrate, NMA-MGPH*FFPK(DNP)dRdR (where an asterisk indicates the scissile site), the standard assay mixture contained varying amounts of the substrate, 50 mm sodium acetate buffer (pH 4.0), and the enzyme in a final volume of 300 μl. The stock enzyme solution used for Method II contained 0.1% (w/v) Tween 80. The assay mixture without the enzyme was brought to 40 °C, and the reaction was started by the addition of the enzyme. After incubation for 10 min, the reaction was stopped by the addition of 300 μl of 1 m Tris-HCl, pH 9.0, followed by chilling the mixture on ice. Fluorescence intensity changes of the reaction mixture (excitation, 340 nm; emission, 440 nm) were determined with a Shimadzu fluorescence spectrophotometer RF-5000. The fluorescence intensity change where the substrate was completely degraded by the addition of an excess amount of the collagenase under these assay conditions was also determined and was used for unit calculations. Protein was determined by the method of Bradford (13Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using a kit (Bio-Rad) with bovine serum albumin as the standard. Kinetic parameters and their S.E. values were determined by nonlinear regression analysis (14Leatherbarrow R.J. Trends Biochem. Sci. 1991; 15: 455-458Abstract Full Text PDF Scopus (172) Google Scholar) using the initial velocity data obtained by means of assay method II. To analyze the P1 and P1′ specificities of kumamolisin-As, peptide libraries were designed on the basis of the amino acid sequence of a nonapeptide, Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser, where an asterisk indicates the scissile site. A peptide of such a sequence was previously identified as a good substrate for kumamolisin-As (4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar). The P1′ library was an equimolar mixture of peptide components, Met-Gly-Pro-Arg*Xaa-Phe-Pro-Gly-Ser, with Xaa being all of the different amino acids other than Cys. The individual relative retention times of the peptide components and their cleavage products were predicted from their amino acid sequences by Rekker's method as described by Sasagawa and Teller (15Sasagawa T. Teller D.C. Hancock W.S. Handbook of HPLC for the Separation of Amino Acids, Peptides and Proteins. CRC Press, Inc., Boca Raton, FL1987: 53-65Google Scholar). Five different sets of peptides (termed P1′a, P1′b, P1′c, P1′d, and P1′e; see below), each consisting of three or four peptides, were synthesized for the P1′ library using an ABI 433A automated peptide synthesizer; the combination of amino acids (Xaa) at P1′ of peptides for each set was determined on the basis of the predicted retention times so that all of the substrate peptide and the cleavage products could be separated from one another by a single HPLC assay (see below). Thus, amino acid residues at the P1′ position of each set were as follows: Trp, His, Gly, and Arg for P1′a; Phe, Leu, Pro, and Ala for P1′b; Tyr, Thr, and Glu for P1′c; Ile, Val, Gln, and Asp for P1′d; and Met, Ser, Lys, and Asn for P1′e. For the evaluation of the P1 specificity of the enzyme, the P1 library, Met-Gly-Pro-Xaa*Phe-Phe-Pro-Gly-Ser, was also constructed in a manner similar to the one described above. Three different sets of peptides were synthesized for the P1 library and were termed P1a (Xaa; His, Asn, Tyr, and Trp), P1b (Xaa; Arg, Glu, Ala, Asp, Val, Leu, and Phe), and P1c (Xaa; Lys, Thr, Gln, Gly, Ser, Met, Pro, and Ile). Primary structures of the peptide libraries were verified by automated Edman degradation. The individual substrates and cleavage products separated on HPLC were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using a Bruker REFLEX III spectrometer. The reaction mixture consisted of 100 mm sodium acetate, pH 4.0, a set of synthetic peptides (typically 250 μm for each component), and 0.185 nm of enzyme in a final volume of 200 μl. The mixture without the enzyme was preincubated at 60 °C, and the reaction was started by the addition of the enzyme. At intervals, a 200-μl aliquot of solution was withdrawn and mixed with 50 μl of 1 m potassium phosphate, pH 7.0; the resulting mixture was chilled on ice. The peptides were analyzed by a reversed-phase HPLC using an automated Gilson 305 system equipped with a Shimadzu SPD-10A VP UV-visible detector: column, YMC-Pack ODS-A A-303 (4.6 × 250 mm; YMC Co., Kyoto, Japan); flow rate, 0.7 ml/min; solvent A, 0.1% (v/v) trifluoroacetic acid; solvent B, 0.1% (v/v) trifluoroacetic acid in 60% (v/v) acetonitrile. After injection (50 μl) onto a column that was equilibrated with 35% solvent B, the column was initially developed isocratically for 5 min, followed by linear gradients from 35% solvent B to 48% solvent B in 20 min and from 48% solvent B to 100% solvent B in 1 min. The column was then washed isocratically with 100% solvent B for 5 min, followed by a linear gradient from 100% B to 35% B in 1 min. This gradient profile ensured base-line separation of the oligopeptide components. The chromatograms were obtained with detection at 215 nm, and the amounts of peptides were determined from peak integrals by using a Shimadzu Chromatopak CR8A data processor. Three independent digestions were carried out to check the reproducibility of the assays. MALDI-TOF mass spectrometry analysis showed that cleavage of the peptides took place only at the predicted site, indicated above by an asterisk. The initial velocity (v) (i.e. change of the individual substrate concentration at zero time) was calculated from the initial linear part of the decrease in the corresponding substrate peak areas after separation of the components by reversed-phase HPLC. Quantitative evaluation of the specificity profiles of the enzyme was based on the equation that describes the enzymatic reaction when competing substrates are present (16Antal J. Pal G. Asboth B. Buzas Z. Patthy A. Graf L. Anal. Biochem. 2001; 288: 156-167Crossref PubMed Scopus (32) Google Scholar, 17Cornish-Bowden A. Fundamentals of Enzyme Kinetics. Portland Press, London1995: 105-111Google Scholar), (vi/[Si])/(vj/[Sj])=(kcat,i/Km,i)/(kcat,j/Km,j)(Eq. 1) where i and j denote the individual substrates, and kcat and Km are the catalytic constant and the Michaelis constant, respectively. Individual concentrations of the substrates, [Si], were determined by amino acid analyses. We constructed an active site mutant of kumamolisin-As in which Glu78 was replaced by His. The plasmid, pScpA/E78H, was prepared by in vitro mutagenesis of the plasmid pScpA (4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar) using a QuikChange™ mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's guidelines. The mutation was verified by DNA sequencing on both strands using a Dye-Terminator Cycle Sequencing Kit (Beckman Coulter, Fullerton, CA) with a CEQ 2000 DNA analysis system (Beckman Coulter). Each of the plasmids, pScpA and pScpA/E78H, was used to transform Escherichia coli BL21 (DE3), and the transformant cells were grown at 37 °C in an LB medium (1 liter) containing 50 μg/ml ampicillin until the optical turbidity of the culture reached 0.6 at 600 nm. Expression of the wild-type and mutated kumamolisin-As genes was attained by adding isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.8 mm, followed by further fermentation for 3 h. The cells were harvested by centrifugation (5,000 × g, at 4 °C for 10 min), suspended in an appropriate volume of a 0.05 m sodium acetate buffer, pH 4.0, and disrupted at 4 °C by ultrasonication at 10 kHz. The cell debris was removed by centrifugation (18,000 × g, at 4 °C for 10 min), and the resultant supernatant (pH 4.0) was incubated at 55 °C for 5 h, followed by centrifugation. Almost all of the endogenous E. coli proteins were removed by centrifugation, and the resultant supernatant contained the expressed product of >96% homogeneity. The supernatant was concentrated by ultrafiltration with a YM-10 membrane using an Amicon 8200 unit and dialyzed at 4 °C against 0.02 m potassium phosphate buffer, pH 7.5 (buffer A). The concentrate was then loaded on an ΔKTA system equipped with a Mono Q HR10/10 column (Amersham Biosciences) equilibrated with buffer A. After loading the enzyme solution onto the column, followed by an extensive washing of the column with buffer A, the enzyme was eluted with a linear gradient of 0.15-0.5 m NaCl in buffer A in 75 min at a flow rate of 0.5 ml/min. In order to avoid any possible contamination of the mutant preparations with the wild-type activity, chromatographic purification of the E78H mutant was completed first, followed by that of the wild-type enzyme. Collagen (3 mg) was reacted with 0.065 pm kumamolisin-As at 60 °C for 30 min in 0.5 ml of a 0.05 m sodium acetate buffer, pH 4.0. The mixture was then analyzed for proteolytic cleavage of collagen by SDS-PAGE (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Protein bands in the gel were transferred to the polyvinylidene difluoride membrane by electroblotting, and the membrane was stained with Coomassie Brilliant Blue R250. Stained portions corresponding to the 70- and 30-kDa fragments of the membrane were excised using dissecting scissors and subjected to automated Edman degradation to determine the N-terminal amino acid sequences of the fragments. Crystals of kumamolisin-As complexed with AcIPF, an inhibitor specifically designed for kumamolisin (10Comellas-Bigler M. Fuentes-Prior P. Maskos K. Huber R. Oyama H. Uchida K. Dunn B.M. Oda K. Bode W. Structure. 2002; 10: 865-876Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), as well as of uninhibited native kumamolisin-As and its E78H mutant were obtained by the vapor diffusion method. The complex was prepared by mixing 100 μl of 2.3 mg/ml kumamolisin-As in 10 mm sodium acetate buffer at pH 5.0 with 2.5 μl of 10 mg/ml AcIPF in Me2SO. The samples of native kumamolisin, both complexed and uninhibited, were mixed with the well solution containing 27% polyethylene glycol 8000, 0.18 m ammonium sulfate, 10 mm dithiothreitol in 25 mm sodium acetate buffer at pH 4.0 or 4.2 at a 1:1 ratio, with the total volume of 4 μl. Crystals of the E78H mutant were grown in a mother liquor containing 0.2 m ammonium sulfate, 30% polyethylene glycol 8000 in deionized water. X-ray diffraction data for the wild-type and E78H mutant apoenzymes were collected on a MAR345 detector mounted on a Rigaku RU200 rotating anode x-ray generator, operated at 50 kV and 100 mA. Data for the inhibitor complex were collected on beamline X9B, NSLS, Brookhaven National Laboratory, using an ADSC Quantum4 CCD detector. The reflections were integrated and merged using the HKL2000 suite (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), with the results summarized in Table I. All structures were refined using the program SHELXL (20Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1892) Google Scholar). After each round of refinement, the models were compared with the respective electron density maps and modified using the interactive graphics display program O (21Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (505) Google Scholar). The default SHELXL restraints were used for the geometrical (22Engh R. Huber R. Acta Crystallogr. A. 1991; 47: 392-400Crossref Scopus (2548) Google Scholar) and displacement parameters; temperature factors were refined isotropically, due to the limited resolution of data. Water oxygen atoms were refined with unit occupancies, although some of the sites are probably only partially occupied. The refinement results are also presented in Table I. The coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1sn7, 1siu, and 1sio for the wild-type apoenzyme, E78H mutant, and the inhibitor complex, respectively).Table IDetails of X-ray data collection and structure refinement.ParametersValuesWild typeE78HWild type/AcIPFSpace groupP1P1P21Unit cell dimensions (Å)a41.8142.1649.37b44.9545.04238.73c49.1149.4349.25α113.9114.8290.0β106.1106.36113.7γ102.3102.0390.0Resolution (Å)2.02.31.8Measured reflections28,81523,703328,423Rmerge (%)3.5 (12.5)aValues in the highest resolution shell are shown in parentheses.4.3 (11.8)5.3 (31.1)I/σ(I)25.8 (6.7)17 (6.1)23 (2.9)Completeness (%)90 (61.6)94.9 (80.6)97 (76)RefinementR, no σ cut-off (%)15.015.917.3Rfree (%)25.528.724.3Reflections used in refinement16,73211,50984,178Reflections used for Rfree8866074,457Root mean square bond lengths (Å)0.0100.0080.011Root mean square angle distances (Å)0.0330.0300.032Protein atoms252625277583Inhibitor atoms87Other ligand atomsbOne Ca2+ ion is present in all molecules; one sulfate ion is bound to each enzyme molecule in the inhibitor complex.1118Water sites286179935Protein Data Bank accession code1sn71siu1sioa Values in the highest resolution shell are shown in parentheses.b One Ca2+ ion is present in all molecules; one sulfate ion is bound to each enzyme molecule in the inhibitor complex. Open table in a new tab Specificity Analysis of Kumamolisin-As—We have previously shown that digestion of collagen with kumamolisin-As at a substrate/enzyme ratio of 103:1 (mol/mol) yielded more than 50 peptides (4Tsuruoka N. Nakayama T. Ashida M. Hemmi H. Nakao M. Minakata H. Oyama H. Oda K. Nishino T. Appl. Environ. Microbiol. 2003; 69: 162-169Crossref PubMed Scopus (55) Google Scholar). Analysis of the primary structure of some of these peptides suggested that the enzyme may preferably act on the -Pro-Xaa*Gly-Yaa-Zaa- sequence of the α1 and α2 chains of collagen. When collagen was incubated with a lower amount of the enzyme (i.e. substrate to enzyme ratio, 2 × 105:1 mol/mol), incubation resulted in specific cleavage of collagen at a single site to produce two degradation products, one with a molecular mass of 70 kDa and another with a molecular mass of 30 kDa, as analyzed by SDS-PAGE. After electroblotting the protein bands of the fragments to the membranes, N-terminal amino acid sequences were analyzed by automated Edman degradation. The N-terminal sequence, NH2-Gly-Leu-Hyp-Gly-Glu-Arg-Gly-Arg-Hyp-, could only be unambiguously determined for the 70-kDa fragment. A comparison of this sequence with the published amino acid sequence of the α1 chain of bovine type I collagen (23Glanville R.W. Breitkreutz D. Meitinger M. Fietzek P.P. Biochem. J. 1983; 215: 183-189Crossref PubMed Scopus (11) Google Scholar, 24Miller E.J. Piez K.A. Reddi A.H. Extracellular Matrix Biochemistry. Elsevier Science Publishing, New York1984: 41-81Google Scholar) revealed that it corresponded to the sequence starting from position 127 of the α1 peptide. Since the N terminus of the α1 chain of bovine type I collagen is blocked (24Miller E.J. Piez K.A. Reddi A.H. Extracellular Matrix Biochemistry. Elsevier Science Publishing, New York1984: 41-81Google Scholar), these results showed that the specific cleavage site of collagen under these conditions was -Arg126-*Gly127- of the α1 collagen chain. The specificity at the P1′ site was then analyzed using the peptide libraries, Met-Gly-Pro-Arg*Xaa-Phe-Pro-Gly-Ser, in which the individual components differ only at the P1′ site. Each set of peptides (see "Experimental Procedures") was digested with kumamolisin-As, and the reaction was monitored by separation of the substrates and cleavage products by reversed-phase HPLC. The calculated specificity profile at the P1′ site is presented in Fig. 1A, indicating some preference for aromatic or bulky aliphatic amino acids. Unexpectedly, Gly was one of the least preferred amino acids at this position, although that residue is present at the P1′ position of the preferred cleavage site in collagen. We then analyzed the P1 specificity profile using the peptide library, Met-Gly-Pro-Xaa*Phe-Phe-Pro-Gly-Ser, where Phe was located at the P1′ site. We found that the P1 site had very high specificity for Arg (Fig. 1B), in excellent agreement with the results obtained by the limited proteolysis of collagen. On the basis of the preliminary results of the specificity studies, we designed an IQF substrate, NMA-MGPH*FFPK-(DNP)DRDR, which could be utilized for a highly sensitive fluorometric assay of kumamolisin-As. This substrate was developed based on the addition of the fluorescent tag, N-methylanthranilic acid (NMA), and the quenching tag, 2,4-dinitrophenol (DNP), to a peptide where His and Phe were located at the respective P1 and P1′ positions. The design was based on our preliminary analysis of the specificity of kumamolisin-As that appeared to indicate that both histidine and arginine were equally good P1 substituents. Although later reinterpretation of the data summarized in Fig. 1B has shown this not to be the case, P1 His appears to be sufficient to provide a basis for the design of an acceptable substrate. Two d-arginine residues were added to the C terminus
Journal Article A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31 Get access Sawao Murao, Sawao Murao Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Yuji Hinode, Yuji Hinode Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Eiko Matsumura, Eiko Matsumura Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, JapanOsaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Search for other works by this author on: Oxford Academic Google Scholar Atushi Numata, Atushi Numata Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, JapanOsaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Search for other works by this author on: Oxford Academic Google Scholar Kenzo Kawai, Kenzo Kawai Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, JapanOsaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Search for other works by this author on: Oxford Academic Google Scholar Hirofumi Ohishi, Hirofumi Ohishi Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, JapanOsaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Search for other works by this author on: Oxford Academic Google Scholar Hisanori Jin, Hisanori Jin Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Hiroshi Oyama, Hiroshi Oyama Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Takashi Shin Takashi Shin Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan Search for other works by this author on: Oxford Academic Google Scholar Bioscience, Biotechnology, and Biochemistry, Volume 56, Issue 6, 1 January 1992, Pages 987–988, https://doi.org/10.1271/bbb.56.987 Published: 01 January 1992 Article history Received: 06 December 1991 Published: 01 January 1992
Experience in three pontine hemorrhage cases which were treated by open surgery is recorded here. They were operated on by trans-fourth ventricular, subtemporal transtentorial on retromastoid suboccipital approaches. The neurological symptom improved noticeably in one case who was operated on through the retromastoid suboccipital approach seven days after the onset. The pontine hemorrhage can even be cured with open surgery which is performed in the early stage and through the operative approach.
