Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds

Rydén, Ann-Christine; Phllipson, Lennart; Rydén, Lars

doi:10.1111/j.1432-1033.1974.tb03462.x

Cited by 45 publications

(15 citation statements)

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“…The enzyme, having a molecular weight of 12,000, is inhibited by diisopropyl fluorophosphate (DFP) but not by EDTA. Similar serine proteases with the same cleavage preference are also found in other S. aureus strains; however, they differ from the above-described enzyme in their size and sensitivity to DFP (2,24). The amino acid sequence of a 27-kDa serine protease of S. aureus revealed a 43-residue C-terminal peptide composed nearly exclusively of aspartic acid, asparagine, and proline (6), proposed to be involved in the transport of the enzyme through the cytoplasmic membrane (7).…”

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confidence: 78%

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Teufel

Götz

1993

J Bacteriol

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The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metallopro- carnosus(pT181mcsPl) corresponded to the extracellular S. epidermidis T03298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc-and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Baciflus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.Most Staphylococcus aureus strains secrete proteolytic enzymes; three different types of proteases have been studied in detail. Serine protease from S. aureus V8, commonly referred to as V-8 protease or protease I, is an endoprotease which cleaves peptide bonds on the carboxy-terminal side of glutamic acid and to a lesser extent of aspartic acid (8). The enzyme, having a molecular weight of 12,000, is inhibited by diisopropyl fluorophosphate (DFP) but not by EDTA. Similar serine proteases with the same cleavage preference are also found in other S. aureus strains; however, they differ from the above-described enzyme in their size and sensitivity to DFP (2, 24). The amino acid sequence of a 27-kDa serine protease of S. aureus revealed a 43-residue C-terminal peptide composed nearly exclusively of aspartic acid, asparagine, and proline (6), proposed to be involved in the transport of the enzyme through the cytoplasmic membrane (7).The second type of protease from S. aureus V8, thiol protease (protease II), is characterized by an isoelectric point of pH 9.4, a molecular weight of 12,500, and a very broad specificity; it also exhibits esterase activity with N-benzoyl-L-tyrosine ethyl ester as a substrate (2). The enzyme is active only in the presence of reducing agents and is inactivated by heavy metals such as Hg2+, Zn2+, or Ag', indicating that protease II must possess free SH groups to be active and that it is a cysteine enzyme.The third protease of S. aureus V8, a metalloprotease (2), is completely inactivated by EDTA and is insensitive to sulfhydryl reagents and DFP. Calcium is essential for the stability of the protease. The molecular weight is 28,000, and the pH optimum for hydrolysis of casein is 7.4. Like the thiol * Corresponding author.protease, the metalloprotease has a rather broad proteolytic specificity, cleaving preferentially at the N-terminal side of hydrophobic residues (1). A metalloprotease with a molecular weight of 38,000 and a pH optimum of 7.0 has been isolated from a mut...

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confidence: 78%

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Teufel

Götz

1993

J Bacteriol

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“…Peptide E contains six dicarboxylic residues apart from carboxymethylcysteine and three of these occur consecutively ( Table 4). The staphylococcal protease was also in this case useful due to its specificity for glutamyl bonds [37,38]. Peptide F (and G) failed to reveal a distinct N-terminus by the dansyl method, although threonine was obtained in low yield.…”

Section: Pep Tide Mappingmentioning

confidence: 99%

“…The dansyl-Edman method was used for sequence analysis of the intact peptides, and of smaller fragments, obtained by digestions with chymotrypsin or a staphylococcal protease preferentially cleaving at glutamyl bonds [37,38]. The results of sequence analysis are given in Table 4, in which secondarily produced fragments are also shown.…”

Section: Pep Tide Mappingmentioning

confidence: 99%

Structural Studies of Adenovirus Type‐2 Hexon Protein

Jörnvall

Pettersson

Philipson

1974

European Journal of Biochemistry

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The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2-14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical.Structural analysis of ['4C]carboxymethylated hexon and of hexon which was labeled in vivo with [3SS]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments.Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained.The adenoviruses are medium-sized icosahedral DNA viruses, some of which are oncogenic. The hexon is the main capsomer, occurring at 240 non-vertex positions in the capsid and contains one of at least ten [1,2] viron polypeptides. It was the first animalvirus protein to be crystallized [3]. It is soluble in native form and is produced in excess during viral infection. Widely different molecular weights have been reported for the intact hexon as well as for its subunits (mainly within values of 200 000 -400 000, and 60000-120000, respectively, for a review, see [4]).The hexon subunit thus contains a large polypeptide, the maximum value of which accounts for the coding capacity of about 11 % of the entire viral genome.Recent analysis of sedimentation velocity in the absence or presence of guanidine-HC1, of equilibrium sedimentation, of crystallographic parameters, andAbbreviations. Tos-PheCH,Cl, L-l-tosylamido-2-phenylethyl chloromethyl ketone; Tos-LysCH,Cl, l-chloro-3-tosylamido-7-amino-2-heptanone; dansyl, S-dimethylaminonaphthalene-l-sulphonyl.Enzymes. Chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4); thermolysin (EC 3.4.24.4); staphylococcal protease (EC 3.4.99 [1,6,7] for the subunit. Crystallographic studies also reveal three asymmetric units in the hexon [4,8], which is consistent with results from electron microscopy [9]. Crystallographic data, however, also suggest additional elements of partial two-fold symmetry [lo] (and P.-E. Werner, personal communication) and an earlier ...

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“…because S. aureus possesses a potent protease (staphylococcal protease) (3,17) which is not inhibited by EDTA. Sonication was carried out to enhance the disruption of cells in the viscous extract (DNA-lysed cell wall complex).…”

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confidence: 99%

DNA gyrase of Staphylococcus aureus and inhibitory effect of quinolones on its activity

Takahata

Nishino

1988

Antimicrob Agents Chemother

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DNA gyrase from Staphylococcus aureus FDA 209P was partially purified by lysostaphin treatment, saturation with ammonium sulfate, and affinity chromatography on heparin-Sepharose and with a concentrator (Centricon 30). It was found to consist of two subunits: a and P. The ability of new quinolone antibacterial agents such as norfloxacin, ofloxacin, and ciprofioxacin to inhibit DNA gyrase activity and cell growth was investigated. The inhibitory effects of the new quinolones against the activity of S. aureus DNA gyrase were in parallel with their antibacterial activities. The 50% inhibitory doses of norfloxacin, ofloxacin, and ciplofloxacin were 0.34, 0.31, and 0.24 ,Ig/ml, respectively, while the 50% inhibitory doses of nalidixic acid and cinmoxacin, which were less active against S. aureus FDA 209P, were 100 ,Lg/ml or more.The DNA gyrases are topoisomerases which catalyze the supercoiling of relaxed covalently closed circular DNA, which is known to be coupled with the hydrolysis of ATP. The enzyme from Escherichia coli (topoisomerase II) is composed of two different subunits, A and B, which are encoded by the genes gyrA and gyrB, respectively (5, 7). The gyrA protein constitutes the target for nalidixic acid and oxolinic acid, and the gyrB protein is the target for novobiocin and coumermycin Al (6,8,9). The target of new quinolones, i.e., norfloxacin, ofloxacin, ciprofloxacin, and enoxacin, is subunit A; and these drugs exhibit marked inhibitory effects (18,21). Evidence has been provided that other gram-negative bacteria possess DNA gyrase and that it is inhibited by quinolones (14,19). In E. coli the active gyrase holoenzyme is a tetramer composed of two subunits, A2 and B2, which have molecular weights of 105,000 and 95,000, respectively (7). In regard to gram-positive bacteria, Orr and Staudenbauer (16) and Sugino and Bott (23) have reported the isolation of a gyrase from Bacillus subtilis, and Liu and Wang (12) have reported the isolation of a gyrase from Micrococcus luteus. Like E. coli, the M. luteus gyrase is a tetramer composed of two subunits, A and B, which have molecular weights of 115,000 and 87,000, respectively. The gyrases from these gram-positive bacteria catalyze the supercoiling of pBR322 DNA or ColEl DNA derived from E. coli (24). New quinolone derivatives such as norfloxacin, enoxacin, ofloxacin, ciprofloxacin, and fleroxacin with a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria have been developed recently (1,10,11,15,20 ATCC 25923 DNA gyrase and inhibition by quinolones, the newer and potent quinolones norfloxacin, ciplofloxacin, and ofloxacin (MICs, <2.0 ,ug/ml) do not inhibit supercoiling (50% inhibitory concentrations, ca. >200 , [g/ml). In this report, we describe the purification of DNA gyrase from S. aureus FDA 209P and the inhibitory effect of new quinolones on its activity. MATERIALS AND METHODSBacterial strain and culture media. S. aureus FDA 209P, which is preserved at the Department of Microbiology, Kyoto Pharmaceutical University, Kyo...

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Isolation and Properties of a Staphylococcal Protease, Preferentially Cleaving Glutamoyl‐Peptide Bonds

Cited by 45 publications

References 30 publications

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Structural Studies of Adenovirus Type‐2 Hexon Protein

DNA gyrase of Staphylococcus aureus and inhibitory effect of quinolones on its activity

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