Harold E. Van Wart

A continuous spectrophotometric assay for Clostridium histolyticum collagenase

Abstract A continuously recording spectrophotometric assay has been developed for Clostridium histolyticum collagenase based on the hydrolysis of 2-furanacryloyl- l -leucylglycyl- l -prolyl- l -alanine (FALGPA). The hydrolysis of this peptide by collagenase obeys Michaelis-Menten kinetics with V = 1.8 × 105μkatal/kg and Km = 0.5 m m . FALGPA is hydrolyzed more rapidly by collagenase than any other commonly used synthetic substrate, but is not cleaved by any of the well-known proteinases such as trypsin, thermolysin, or elastase. The assay itself is rapid, convenient, and sensitive, and should greatly facilitate detailed kinetic studies of collagenase.

Continuously recording fluorescent assays optimized for five human matrix metalloproteinases

Four new fluorogenic heptapeptide substrates have been synthesized with sequences that are optimized for five human matrix metalloproteinases (MMP). All four substrates are similar to one recently reported by Stack and Gray (1989, J. Biol. Chem. 264, 4277-4281) and have the fluorescent Trp residue in subsite P2 and the dinitrophenol (DNP) quenching group on the N-terminus. The quenching of the Trp fluorescence in the intact substrate is relieved on hydrolysis of the P1-P1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P1 and P3 have been optimized for each MMP, while Arg has been placed in P4 to enhance solubility. Thus, DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg has been prepared as a substrate for fibroblast collagenase, DNP-Pro-Leu-Ala-Tyr-Trp-Ala-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for stromelysin, and DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg for both 72-kDa fibroblast gelatinase and 92-kDa neutrophil gelatinase. These substrates have been characterized with respect to their composition, solubility, optical and fluorescence spectra, and hydrolysis by their target MMP. The hydrolysis rates rival or exceed those of either their natural protein substrates or other synthetic peptides. The solubility of each substrate in assay buffer exceeds the KM value for each reaction, allowing accurate determination of the kinetic parameters. These new substrates should greatly facilitate kinetic studies of the MMP.

PROTEOLYTIC AND NON-PROTEOLYTIC ACTIVATION OF HUMAN NEUTROPHIL PROGELATINASE B

The activation of human neutrophil progelatinase B (pro-HNG) by a variety of proteolytic and non-proteolytic activators has been investigated. A quantitative comparison of the activation efficiencies of treatments previously reported to activate pro-HNG or the related gelatinase B species produced by other cells demonstrates that stromelysin and trypsin are good activators. HgCl2 is a moderately effective activator, while p-chloromercuribenzoate and NaOCl are poor activators. It is also shown that human matrilysin and human fibroblast-type collagenase can activate pro-HNG by a mechanism that is very similar to that of stromelysin. Initially, these proteinases hydrolyze the Glu40-Met41 bond in the propeptide domain to generate an 88 kDa inactive HNG species. Collagenase also generates a 68 kDa HNG species through hydrolysis of the Ala74-Met75 bond. Ultimately, treatment with either matrilysin, collagenase or trypsin results in the production of a 65 kDa active form of HNG that arises from hydrolysis of the Arg87-Phe88 bond. This is the same active species produced on activation by stromelysin. This cleavage site is downstream of the cysteine-switch residue located at position 80 and releases it, accounting for the permanent activation of the enzyme. These results suggest that matrilysin and collagenase may be physiologically relevant activators of pro-HNG and/or other progelatinase B species. Activation by HgCl2 produces an active 68 kDa enzyme due to autolytic hydrolysis of the Ala74-Met75 bond. This species retains the cysteine switch residue; however, it is shown that it is only active in the continued presence of HgCl2. Removal of the HgCl2 restores latency, indicating that this species is reversibly activated by HgCl2, which functions by complexing the sulfhydryl group of the cysteine switch residue and keeping it dissociated from the active site zinc atom. Thus, in spite of reports to the contrary, the cysteine switch mechanism can account for the latency and activation of pro-HNG.

Synthetic inhibitors of bacterial and mammalian interstitial collagenases.

Publisher Summary This chapter reviews recent progress in the development of both bacterial and mammalian collagenase inhibitors. To facilitate this goal, one provides relevant biochemical background information on these enzymes and their assay methods. The chapter also discusses the many roles that these collagenases are believed to play in both normal and pathological processes. The chapter summarizes the inhibitors that have been developed and the results on the use of a few collagenase inhibitors in physiological assays. The extracellular matrix in humans is composed of a variety of macromolecules that serve both structural and organizational roles. These include collagens, elastin, proteoglycans, fibronectin, laminin, and other poorly characterized minor substituents. The collagens account for about one-third of human protein and are the primary components of most connective tissues including skin, bone, teeth, tendon, cartilage, basement membrane, blood vessels, cornea and the vitreous humor. They are ubiquitous in higher organisms, in which they occur as aggregates and function as structural proteins. The term collagen actually refers to a class of molecules derived from one or several multigene families.

High-valent intermediates in the reaction of Nα-acetyl microperoxidase-8 with hydrogen peroxide: Models for compounds 0,I and II of horseradish peroxidase

N-acetyl microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for the peroxidases. The reaction of Ac-MP-8 with H2O2 in 10 mM potassium phosphate over the pH range of 7-11 gives rise sequentially to relatively stable green and red species with properties that closely mimic those of HRP compounds I and II, respectively. Low-temperature stopped-flow studies of this reaction carried out in 50% v/v methanol/10 mM potassium phosphate, pH* 9.1 at -25.8 degrees C indicate that the pseudo-first-order rate constant, kobs, that describes the formation of the green intermediate exhibits saturation kinetics as a function of [H2O2] with kmaxobs = 95 s-1 and KM = 87 mM. Rapid-scan studies carried out with [H2O2] = 200 mM at -38.0 degrees C show that a compound 0 species with a characteristic band near 340 nm is formed whose conversion to the green species is rate limiting. Thus, Ac-MP-8 has high-valent forms that are models for all three known intermediates in the peroxidase cycle of horseradish peroxidase.

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4°c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k 1) was 2.3 × 107 m −1 s−1 for PGHS-1 and 2.5 × 107 m −1s−1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k 2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 102–103 s−1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that thek 2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.

Identification of Clostridium histolyticum Collagenase Hyperreactive Sites in Type I, II, and III Collagens: Lack of Correlation with Local Triple Helical Stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.

Kinetics of hydrolysis of type I, II, and III collagens by the class I and IIClostridium histolyticum collagenases

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [3H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parameterskcat andKM have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parameterskcat andKM for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.

Adult human endothelial cell enzymatic harvesting: Estimates of efficiency and comparison of crude and partially purified bacterial collagenase preparations by replicate microwell culture and fibronectin degradation measured by enzyme-linked immunosorbent assay

Reliable enzymatic endothelial cell (EC) harvest methods are required for clinical EC seeding of vascular prostheses by methods analogous to those demonstrated in dogs. But crude collagenases used for EC harvest vary in efficacy and cytotoxicity, and purified collagenases reportedly give low EC yields. To compare different harvest methods, we studied growth curves of primary adult human saphenous vein EC (HSVEC) harvests plated in replicate microwell cultures. The EC yield, defined as attachment-capable ECs obtained per square centimeter of vein lumen, was estimated from the lowest number of ECs counted in lag phase before exponential growth began. With the use of morphometric studies of HSVs that were perfusion-fixed at their original dimensions, the baseline in situ density of ECs available for harvest from HSV was estimated at 1.3 X 10(5) EC/cm2. Crude (CBC) and partially purified bacterial collagenase (PBC) solutions at concentrations with equal levels of basement membrane lysis activity (BMLA) were compared by the replicate microwell method in a series of 21 harvests (six CBC, eight PBC, and seven enzyme-free control harvests). All 14 enzymatic harvests produced confluent EC cultures with no significant difference in mean harvest efficiency between CBC (12% of in situ EC number) and PBC (15%). However, PBC caused less degradation of human fibronectin (p less than 0.0001) as measured by an enzyme-linked immunosorbent assay employing a fibronectin-specific monoclonal antibody. These data suggest that chemically defined mixtures of pure enzymes with BMLA equal to the BMLA of crude collagenase might allow reliable EC harvesting without sacrifice in EC yield but with improved preservation of structures at the EC periphery. EC losses during initial vein dissection may have contributed to the low 12% to 15% efficiency we observed.

Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III 3H-acetylated collagens by bacterial and tissue collagenases

Accurate and quantitative assays for the hydrolysis of soluble 3H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30 degrees C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations. that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters Km and kcat or Vmax. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.