Inactivation of creatine kinase induced by dopa and dopamine in the presence of ferrylmyoglobin

We investigated the effect of dopa and dopamine on creatine kinase (CK) activity in the presence of ferrylmyoglobin (ferrylMb). CK was sharply inhibited by dopa and dopamine in the presence of ferrylMb. Dopa and dopamine markedly promoted the reduction of ferrylMb to metmyoglobin (metMb). The semiquinone from dopa and dopamine may be involved in CK inactivation. During inactivation of the enzyme, both kinetic parameters Vmax and Km changed. In addition, reduced glutathione restored the activity of CK at an early stage. These results suggest that inactivation of CK is dominantly due to oxidation of sulfhydryl (SH) groups of the enzyme. Other catechols, such as adrenaline and noradrenaline, little inactivated CK activity, whereas they promoted the reduction of ferrylMb to metMb. Other SH enzymes, including alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were inactivated to a lesser extent by dopa and dopamine in the presence of ferrylMb. Adrenaline and noradrenaline did not significantly prevent the inactivation of ADH and very slightly inhibited GAPDH. These results suggest that dopa and dopamine act as prooxidants to inactivate SH enzymes in the presence of ferrylMb.     

Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic-AMP-dependent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle

The glycogen-bound form of protein phosphatase-1 (termed protein phosphatase-1G) is composed of the catalytic (C) subunit complexed to a glycogen-binding (G) subunit that anchors the enzyme to glycogen [Str?lfors et al. (1985) Eur. J. Biochem. 149, 295-303]. Incubation of purified protein phosphatase-1G with cyclic-AMP-dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G-subunit [Caudwell et al. (1986) FEBS Lett. 194, 85-90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen-protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase-1 activity associated with the glycogen-protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase-1 from glycogen may facilitate its inactivation by inhibitor-1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase-1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.     

Control of rat skeletal-muscle phosphorylase phosphatase activity by adrenaline.

Administration of adrenaline to an isolated rat hindlimb preparation rapidly decreased muscle phosphorylase phosphatase (EC 3.1.3.17) activity and increased heat-stable and trypsin-labile phosphatase inhibitor activity. This was associated with increased tissue cyclic AMP concentrations, phosphorylase (EC 2.4.1.1) activation and glycogen synthase (EC 2.4.1.11) inactivation.     

Nickel controls the reversible anaerobic activation/inactivation of the Desulfovibrio gigas hydrogenase by the redox potential

An electrochemical method of hydrogenase activity measurement is developed. It permits a new approach to the activation/inactivation process of the Desulfovibrio gigas hydrogenase. A monolayer of hydrogenase is grafted onto a glassy carbon electrode which is both the support of the enzyme and the detector of the activity. The physicochemical composition of the enzyme microenvironment is thus well defined and easily controlled by the electrode potential. Successive periods of inactivation and activation are applied to the same hydrogenase molecules, thus the activity can be correlated to the chronology of the experiments. We distinguish two kinds of activation/inactivation processes. The first one, already described for the enzyme stored for some months in aerobic conditions, is a slow activation by molecular hydrogen or a reducing medium (half-reaction time = 2 h). The second one is an anaerobic inactivation by an oxidizing potential. This first order inactivation (half-reaction time = 10 min) is fully reversible. This modulation of the activity level is controlled by an Ni(III)/Ni(II) redox couple (Eh = -455 mV/calomel-saturated KCl electrode at pH 8.3) involving one electron and one proton. This work proposes an explanation for the activation of the hydrogenase taking into account the participation of an [Fe-S] cluster and of the nickel atom.     

Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice.

In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.     

Effect of urate on the lactoperoxidase catalyzed oxidation of adrenaline

Lactoperoxidase is an iron containing enzyme, which is an essential component of the defense system of mammalian secretary fluids. The enzyme readily oxidizes adrenaline and other catecholamines to coloured aminochrome products. A K_m-value of 1.21 mM and a catalytic constant (k = V_\max/[Enz]) of 15.5 × 10³ min^–1 characterized the reaction between lactoperoxidase and adrenaline at pH 7.4. Urate was found to activate the enzyme catalyzed oxidation of adrenaline in a competitive manner, the effect decreasing with increasing adrenaline concentration. Lactoperoxidase was able to catalyze the oxidation of urate. However, urate was a much poorer substrate than adrenaline, and it seems unlikely that urate activates by functioning as a free, redox cycling intermediate between enzyme and adrenaline. The activation mechanism probably involves an urate-lactoperoxidase complex.     

Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation.

Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.     

Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37-40 degrees C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550-630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35 degrees C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37-40 degrees C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.     

5'-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4?min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic 'metabolic stress'. Inactivation of AMPK by adrenaline was abolished by 1?μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.     

Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins.

Prostaglandin endoperoxide synthetase purified to apparent homogeneity from bovine vesicular gland microsomes contained iron far below the equimolar amount and essentially no heme. However, the enzyme required various metalloporphyrins including hematin or several hemoproteins such as hemoglobin. Preincubation of the enzyme with hematin or hemoglobin resulted in the loss of enzyme activity. The enzyme inactivation was protected by tryptophan or various other aromatic compounds. Furthermore, the simultaneous presence of tryptophan brought about activation of enzyme; namely, the enzyme preincubated with heme and tryptophan showed an almost full activity with a heme concentration in the reaction mixture far below the saturating level. Such inactivation and activation of the enzyme were also observed with manganese protoporphyrin. An identical heme requirement, heme-induced inactivation, and activation of the enzyme were observed in three types of reactions catalyzed by the enzyme: 1) bis-dioxygenation of 8,11,14-eicosatrienoic acid to produce prostaglandin G1, 2) 15-hydroperoxide cleavage of prostaglandin G1 to produce prostaglandin H1, and 3) guaiacol peroxidation. When heme was replaced by manganese protoporphyrin, the enzyme catalyzed only the bis-dioxygenation producing prostaglandin G1 and the activities of the latter two reactions were not detectable.     

Modification of lipoxygenase by hydrogen peroxide and photooxidation.

The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.     

Activation of rat liver branched-chain 2-oxo acid dehydrogenase in vivo by glucagon and adrenaline.

The activity of liver branched-chain 2-oxo acid dehydrogenase complex was measured in rats fed on low-protein diets and given adrenaline, glucagon, insulin or dibutyryl cyclic AMP in vivo. Administration of glucagon or adrenaline (200 micrograms/100 g body wt.) resulted in a 4-fold increase in the percentage of active complex. As with glucagon and adrenaline, treatment of rats with cyclic AMP (5 mg/100 g body wt.) resulted in marked activation of branched-chain 2-oxo acid dehydrogenase. Insulin administration (1 unit/100 g body wt.) also resulted in activation of enzyme; however, these effects were less than those observed with glucagon and adrenaline. In contrast with the results obtained with low-protein-fed rats, administration of adrenaline (200 micrograms/100 g body wt.) to rats fed with an adequate amount of protein resulted in only a modest (14%) increase in the activity of the complex. The extent to which these hormones activate branched-chain 2-oxo acid dehydrogenase appears to be correlated with their ability to stimulate amino acid uptake into liver.     

Activation of sucrose-phosphate synthase from Prosopis juliflora in light: Effects of protein kinase and protein phosphatase inhibitors

Sucrose‐phosphate synthase (SPS) activity measured under limiting substrate and in the presence of inorganic phosphate as an allosteric inhibitor (V_lim activity) from the leaves of Prosopis juliflora was earlier observed to respond rapidly and reversibly to light/dark transitions ( Sinha et al. 1997b,c ). The experiments therefore, were conducted to study the potential regulation of the enzyme by a mechanism of phosphorylation/dephosphorylation. The desalted extract of the enzyme prepared from irradiated leaves showed a time‐dependent spontaneous inactivation of the V_lim activity when the extract was preincubated and an additional inactivation when incubated with ATP. The spontaneous inactivation is not inhibited by phosphatase inhibitors but the ATP‐dependent inactivation was abolished when either 5′‐p‐fluorosulphonylbenzoadenosine (FSBA) or glucose‐6‐phosphate (G6P), (both reported as inhibitors for the SPS‐protein kinase from spinach) was included during preincubation. FSBA also prevented the dark inactivation of SPS in the leaves of P. juliflora when fed through the transpiration stream. The activity of SPS measured under the V_max condition remained relatively unaffected by ATP or FSBA. The desalted extract prepared from darkened leaves on the other hand, when preincubated at 25°C showed a time‐dependent increase in the V_lim activity and the activation state of the enzyme. The spontaneous activation observed during preincubation appears to be due to the dephosphorylation of the enzyme and is strongly inhibited by okadaic acid, a potent protein phosphatase inhibitor. Alternately, feeding okadaic acid to excised leaves in the dark also blocked the subsequent light activation of V_lim activity. These results are consistent with the assumption that the light/dark regulation of V_lim activity observed in the leaves of P. juliflora was mediated through a dephosphorylation/phosphorylation mechanism.     

Trypsin-catalyzed activation of aspartase

Aspartase [EC 4.3.1.1] of Escherichia coli is several-fold activated by treatment with trypsin. The activation requires a few minutes to attain a maximal level, and hereafter the enzyme activity gradually decreases resulting in a complete inactivation in about 4 hours. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. No appreciable change is detected in the molecular weight of the subunits upon trypsin-mediated activation as judged from dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the structural alteration of the enzyme associated with the activation is a minor one. Kinetic properties of aspartase are also compared before and after the trypsin-activation.     

Hormonal activation of type-L hormone-sensitive lipase measured in defatted heart powders.

Adrenaline, 3-isobutyl-1-methylxanthine (MIX) and dibutyryl cyclic AMP (Bt2 cyclic AMP) stimulated type-L hormone-sensitive lipase (HSL) activity when measurements were made on defatted rat heart powders. These lipolytic agents stimulated the activity of this enzyme in a time- and dose-dependent manner. This activation was reversible, because removal of adrenaline from the perfusate was accompanied by the return of type-L HSL activity to control levels. We have reported [Palmer, Caruso & Oscai (1981) Biochem. J. 198, 159-166] that perfusion with low levels of adrenaline, MIX or Bt2 cyclic AMP reduced type-L HSL activity below control levels when measurements were made in aqueous homogenates. However, in the present study, when activities were measured in acetone/diethyl ether heart powders, all concentrations of these agents studied stimulated enzyme activity, and at no concentration was there enzyme inhibition. These data suggest that acetone/diethyl ether treatment may remove a factor that plays a role in type-L HSL regulation. Type-L HSL activity measured in acetone/diethyl ether powders of control and stimulated rat heart exhibited properties that include alkaline pH optimum, serum requirement, activation by heparin and inhibition by high salt and protamine sulphate. These characteristics, in addition to the stability of the enzyme to treatment with organic solvents, fulfil the requirements for the type-L HSL classification.     

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.     

Adrenaline and the regulation of acetyl-coenzyme A carboxylase in rat epididymal adipose tissue. Inactivation of the enzyme is associated with phosphorylation and can be reversed on dephosphorylation.

1. Exposure of rat epididymal fat-pads or isolated fat-cells to adrenaline results in a decrease in acetyl-CoA carboxylase activity measured both in initial extracts and in extracts incubated with potassium citrate; in addition the concentration of citrate required to give half-maximal activation may also be increased. 2. Incorporation of 32Pi into acetyl-CoA carboxylase within intact fat-cells was investigated and evidence is presented that adrenaline increases the extent of phosphorylation of the enzyme. 3. Dephosphorylation of 32P-labelled acetyl-CoA carboxylase was studied in cell extracts. The rate of release of 32P is increased by 5mM-MgCl2 plus 10--100 microM-Ca2+, whereas it is inhibited by the presence of bivalent metal ion chelators such as EDTA and citrate. 4. The effects of adrenaline on the kinetic properties of acetyl-CoA carboxylase disappear if pad or cell extracts are treated with Mg2+ and Ca2+ under conditions that also lead to dephosphorylation of the enzyme. 5. The results of this study represent convincing evidence that adrenaline inactivates acetyl-CoA carboxylase in adipose-tissue preparations by increasing the degree of phosphorylation of the enzyme.     

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTPγS), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t_1.2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca²⁺ ions inhibited the enzyme (half-maximal effect at 0.3 μM), whereas InsP₃ had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells. © 1996 Wiley-Liss, Inc.     

Different effects of hypochlorous acid on human neutrophil metalloproteinases: activation of collagenase and inactivation of collagenase and gelatinase.

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.