Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos.

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.

Mechanism of inhibition of human neutrophil collagenase by Gold(I) chrysotherapeutic compounds. Interaction at a heavy metal binding site.

The mechanism of inhibition of two forms of human neutrophil collagenase (HNC) by six Au(I) compounds, some of which are used as chrysotherapeutic agents, has been investigated. The two forms of enzyme studied are active and latent HNC, the latter of which is activated by p-chloromercuribenzoate (PCMB). The effects of PCMB and Zn(II), which are normally included in the assays, on the activity of both forms of HNC and on their inhibition by these Au(I) compounds have also been studied. Zn(II) stimulates the activity of both the active and PCMB-activated latent forms of HNC up to a concentration of 50-100 microM, after which it inhibits markedly. PCMB activates latent HNC up to a concentration of 100 microM followed by inhibition at higher concentrations. Active HNC is not stimulated at PCMB concentrations below 100 microM, but is inhibited at higher concentrations. The stimulatory effects of Zn(II) and PCMB on HNC and its inhibition by PCMB are all attributable to binding at distinct sites. The inhibition of both active and PCMB-activated latent HNC by the Au(I) compounds is noncompetitive and is reversed by Zn(II). The inhibition of both forms of HNC by SKF 80544 and SKF 36914, which do not contain thiol ligands, is weak to moderate and is not influenced by the PCMB concentration. In contrast, PCMB markedly enhances the inhibition by Myocrisin, Sanocrisin, and Solganol by complexing to their thiol ligands to facilitate release of the Au(I) atom for binding to HNC. Cd(II) and Cu(II) also inhibit HNC noncompetitively, and inhibition is also reversed by Zn(II). Collectively, these data indicate that latent HNC contains a heavy metal binding site distinct from the active site at which Au(I), Cd(II), and Cu(II) bind to cause noncompetitive inhibition. Occupancy of this site by Zn(II) is characterized by retention of activity.

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy.

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium 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 II Clostridium 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 parameters kcat and KM 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 parameters kcat and KM 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.

A sensitive, continuously recording fluorogenic assay for calpain.

We have developed a sensitive and continuously recording fluorogenic assay for the thiol protease calpain. This assay uses the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buffer (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 picomolar calpain making it more sensitive than any previously published method. Moreover, this assay gives a rate that is linear for over ten minutes making it useful for mechanistic studies of inhibitors. This assay can be easily adapted to a 96-well plate format facilitating the large scale screening of inhibitors.

Properties of radiolabeled type I, II, and III collagens related to their use as substrates in collagenase assays.

Calf skin and rat tendon type I, bovine cartilage type II, and human amnion type III collagens have been radiolabeled by reaction with [3H]acetic anhydride, [3H]formaldehyde, and succinimidyl 2,3-[3H]propionate. All three reactions produce collagens with high specific activities that are suitable for use as substrates in collagenase assays. The identity of the radiolabel and the labeling indices do not alter the molecular weights or thermal stabilities of the collagens or the solubilities of the collagens or gelatins in dioxane-water mixtures at 4 degrees C. However, in contrast to native or sparsely labeled collagens, those with 40 or more lysine + hydroxylysine residues labeled per molecule do not undergo fibrillogenesis in the presence of 0.2-0.4 M NaCl in the 4-35 degree C temperature range. Thus, the modification reactions not only serve to introduce the radiolabel, but also to keep the collagens soluble over a wide range of temperatures and concentrations. The TCA, TCB fragments produced on partial reaction of each collagen type with tissue collagenases can be selectively denatured by a 10-minute incubation under specific conditions and the intact collagens selectively precipitated by addition of 50% v/v dioxane. This serves as the basis for soluble collagenase assays. The effect of labeling index on the properties of the collagens has been investigated and the results establish the range of conditions over which these collagens can be used as substrates for soluble versus fibrillar collagenase assays.

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.

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 P'2 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-P'1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P'1 and P'3 have been optimized for each MMP, while Arg has been placed in P'4 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.

Biologically active monomeric and heterodimeric recombinant human calpain I produced using the baculovirus expression system.

Calpain I is a heterodimeric protein that is part of a family of calcium-activated intracellular cysteine proteases presumed to play a role in mediating signals transduced by calcium. Expression of bioactive recombinant human calpain I has been achieved using the baculovirus expression system, by either co-infection with two viruses, each expressing one of the subunits, or infection with a single virus containing both subunits. The approximately 80 kDa catalytic subunit exhibited calcium-dependent proteolytic activity when expressed alone or with the approximately 30 kDa regulatory subunit. Baculoviral recombinant calpain I appeared fully active in that the catalytic subunit in unpurified cell extracts exhibited calcium-dependent autocatalytic cleavage at the correct locus. The amount of approximately 80 kDa subunit accumulated at steady state was greatly increased by co-expression of the approximately 30 kDa subunit, suggesting a possible role for enzyme stabilization by the latter subunit. The recombinant human calpain I was purified to near homogeneity and compared with purified native human erythrocyte calpain I. The recombinant and native enzymes had equivalent inhibition constants for structurally diverse calpain inhibitors, identical calcium activation profiles, and similar specific activities, demonstrating the suitability of using the recombinant protein for studies of the native enzyme.

Inhibition of human collagenases by sulfur-based substrate analogs.

A series of sulfhydryl and novel sulfur-based substrate-analog inhibitors has been synthesized and tested against human fibroblast and neutrophil collagenases. Absolute stereospecific synthesis of several sulfhydryl inhibitors establishes that it is the diastereomers with the R-configuration of the P'1 residues, which correspond to the unnatural D-amino acid analogs, that are the most potent inhibitors. The corresponding disulfide, sulfonate, sulfinate, sulfide, sulfoxide and sulfone analogs exhibit widely variable levels of potency, but all less than the sulfhydryl compounds. No correlation between inhibitor potency and any single structural feature of these new compounds is apparent. However, differences in potency can be ascribed to the different affinities of these functional groups for zinc coordination and hydrogen bonding to nearby active site residues.

Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase.

A series of experiments has been carried out to characterize 58-kDa human neutrophil collagenase (HNC) and compare it with human fibroblast collagenase (HFC). N-Terminal sequencing of latent and spontaneously activated HNC shows that it is a distinct collagenase that is homologous to HFC and other members of the matrix metalloproteinase gene family. Activation occurs autolytically by hydrolysis of an M-L bond at a locus homologous to the Q80-F81-V82-L83 autolytic activation site of HFC. This releases a 16-residue propeptide believed to contain the "cysteine switch" residue required for latency. Polyclonal antibody raised against HNC cross-reacts with HFC but with none of the other major human matrix metalloproteinases examined. Treatment of HNC with endoglycosidase F or N-glycosidase F indicates that it is glycosylated at multiple sites. The deglycosylated latent and spontaneously activated enzymes have molecular weights of approximately 44K and 42K, respectively. Differences in the carbohydrate processing of HFC and HNC may determine why HFC is a secreted protein while HNC is stored in intracellular granules. The kinetic parameters kcat and KM for the hydrolysis of the interstitial collagen types I, II, and III in solution by both collagenases have been determined. The strong preferences of HNC for type I collagen and of HFC for type III collagen found in earlier studies have been confirmed. The preference of HNC for type I over type III collagen is almost abolished when fibrillar collagens are used as substrates, but the preference for HFC for type III over type I collagen is only partially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)