Leu-Gly Bond Research Articles

Purification and Characterization of a Major Collagenase fromStreptomyces parvulus

A major collagenase was purified about 96-fold from a crude enzyme sample of Streptomyces parvulus by chromatography on Q-Sepharose, Sephacryl S-200, and butyl-Toyopearl. The purified enzyme showed a relative molecular mass of approximately 52,000 on SDS-PAGE and a pH optimum at about 9.0, and was strongly inhibited by metal-chelating agents. It also cleaved 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg specifically at the Leu-Gly bond, with a K(m) value of 0.60 mM at pH 9.0 at 37 degrees C. Based on the amino acid sequences of the N-terminal region and internal tryptic peptides, the corresponding gene was cloned. The DNA sequence of the cloned gene indicated that the enzyme is produced as an 864-residue precursor protein with a 408-residue prepro sequence followed by a 456-residue mature enzyme moiety. The enzyme is most homologous with the collagenase from S. coelicolor, the identity being 73%, and it is thought to be a member of the Vibrio collagenase subfamily.

Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin.

An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.

A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid

DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.

Hydrolysis of the Leu-Gly bond of phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-D-Arg (a substrate of microbial collagenases) by treponemes isolated from the subgingival plaque of periodontitis patients

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl-l-prolyl-l-leucylglycyl-l-prolyl-d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US),Treponema denticola ATCC 35405, and 29 different clinical isolates ofT. denticola. TheTreponema US enzyme also hydrolyzed furylacryloyl-l-leucylglycyl-l-prolyl-l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators andp-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by “true” microbial collagenases.

Collagenase-like (CL) peptidase activity in synovial fluid from patients with rheumatoid arthritis

We found the presence of collagenase-like (CL) peptidase in synovial fluid by a highly sensitive fluorescence assay using (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA) as a substrate. Suc-GPLGP-MCA is hydrolyzed at the Leu-Gly bond by CL-peptidase. The CL-peptidase activity in synovial fluid was significantly higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA) and in arthropathy-free controls. No significant difference in CL-peptidase activity in synovial fluid was found between patients with OA and arthropathy-free controls.

Determination of the cleavage specificity of Streptomyces griseus protease B in the presence of guanidinium chloride.

Streptomyces griseus protease B (SGPB), a Pronase enzyme, has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Siegel, S. et al. (1972) J. Biol. Chem. 247, 4155-4159). In order to determine the cleavage specificity of this unusual enzyme under denaturing conditions, 12 peptides of known amino acid sequence were hydrolyzed by SGPB in the absence and presence of 6.0 M guanidinium chloride. The new N-terminal amino acids produced by the action of SGPB were dansylated and quantitatively identified by reverse phase HPLC. The results indicate that SGPB retained its cleavage specificity for phenylalanyl, tyrosyl, tryptophanyl, and leucyl peptide bonds in the presence of 6.0 M guanidinium chloride. Of these peptide bonds, SGPB exhibited a greater cleavage preference for phenylalanyl and tryptophanyl bonds, which was relatively unaffected by the presence of the denaturant. The SGPB-catalyzed cleavages of the leucyl peptide bonds examined (Leu-Met, Leu-Arg, Leu-Val, Leu-Thr, and Leu-Ile) were substantially decreased under denaturing conditions, while Leu-Gly bond cleavage by SGPB was virtually unaffected by denaturant. The demonstrated predictability of the catalytic preference of this unusual protease for phenylalanyl, tyrosyl, tryptophanyl, and leucyl-glycine peptide bonds under denaturing conditions enhances its utility in the site-specific proteolysis of insoluble or otherwise proteolysis-resistant polypeptide substrates.

Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities.

The action of three class I (beta, gamma, and eta) and three class II (delta, epsilon, and zeta) collagenases from Clostridium histolyticum on two series of peptides with collagen-like sequences has been examined. The peptides in the first series all contain 4-nitrophenylalanyl-Gly-Pro-Ala in subsites P1 through P3', but each is successively lengthened in the N-terminal direction by addition of an appropriate residue until subsite P5 is occupied. The second group of peptides all have cinnamoyl-Leu in subsites P2 and P1, respectively, but each is successively lengthened in the C-terminal direction by partial additions of the Gly-Pro-Leu triplet until subsite P6' is occupied. N-Terminal elongation causes the kcat/KM values to rise markedly and to level off after occupancy of subsite P6 for the class I enzymes and subsite P3 for the class II enzymes. C-Terminal elongation produces the best substrates for both classes of enzymes when subsites P3' or P4' are occupied by amino acids with free carboxyl groups. The kcat/KM values for the hydrolysis of both Leu-Gly bonds of cinnamoyl-Leu-Gly-Pro-Leu-Gly-Pro-Leu have been measured for both classes of enzymes. Both rates are large, but both classes preferentially hydrolyze the Leu-Gly bond of the C-terminal triplet. Thus, both classes of enzymes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities.

Simultaneous analyses of clostridium collagenase and neutral proteinase activities with a single synthetic substrate

Two simple methods are presented for simultaneously detecting the activities of both Clostridium histolyticum collagenase and a neutral proteinase that contaminates commercial preparations of collagenase, using a single common substrate carbobenzoxy-Gly-Pro-Leu-Gly-Pro. This pentapeptide is cleaved only once either at the Leu-Gly bond by collagenase or at the Pro-Leu bond by the neutral proteinase. Both products, Gly-Pro and Leu-Gly-Pro, can be analyzed not only semiquantitatively by thin-layer chromatography on cellulose sheets, but also quantitatively by direct application of the digest to an automatic amino aicd analyzer for the calculation of initial velocities. These methods have an advantage over the conventional caseinolysis assay in the time required for incubation. A tetrapeptide, carbobenzoxy-Gly-Pro-Leu-Gly, is also cleaved by the neutral proteinase, but less effectively than the pentapeptide. It is proposed that this proteinase has thermolysin-like specificity.

Separation of collagenase and a metal-dependent endopeptidase of rat uterus that hydrolyzes a heptapeptide related to collagen

1. 1. The synthetic peptide 2,4-dinitrophenyl- l-Pro- l-Leu-Gly l-Ile- l-Ala-Gly- l-Arg-amide (DNP-peptide) was tested as a potential substrate for uterine collagenase. Rat uteri were homogenized and the insoluble fraction was extracted at 60° C to obtain collagenase. The extracts were chromatographed on Sephadex G-150 to yield two peaks of DNP-peptide hydrolyzing activity. Peak I was completey inhibited by EDTA and had a molecular weight greater than 100 000. Peak II was inhibited about 90% by EDTA and had an apparent molecular weight of about 70 000. 2. 2. Peak II coincided closely, but not exactly, with the peak of collagenase activity. It differed from collagenase in heat stability, binding properties on CM-Sephadex and failure to display latency. 3. 3. Peak II represents a new endopeptidase activity. It has a pH optimum of 7 and it cleaves the DNP-peptide at the Gly-Ile and, possibly, the Leu-Gly bond. 4. 4. The DNP-peptide is not a satisfactory substrate for the assay of impure collagenase preparations nor does it inhibit the action of collagenase on collagen substrate when added in 30-fold molar excess.

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity

A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA). Suc-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluorescence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K m value of 4.0 × 10 −4 m toward Suc-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 ± 0.10 nmol/min/ml of serum ( n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro- d-Arg (PZ-peptide). The new peptide, Suc-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.