Comparative kinetic characteristics of catalase of Penicillium species molds

Extracellular catalases produced by fungi of the genus Penicillium: P. piceum, P. varians and P. kapuscinskii were purified by consecutive filtration of culture liquids. The maximum reaction rate of H2O2 decomposition, the Michaelis constants and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by effective rate of catalase inactivation during enzymatic reaction (kin at 30 degrees C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45 degrees C (k*[symbol: see text]H, s-1). Catalase from P. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase from P. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeast C. boidinii.     

Kinetic Characterization of Extracellular Catalases fromPenicillium piceum F-648 and Its Hydrogen Peroxide-Adapted Variants

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H₂O₂ was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H₂O₂ degradation (V _max), V _max/K _M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k _in, s^–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k _in ^*, s^–1) was determined at 45°C. In all samples, the specific activity and K _M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10^–4 mg/ml. The effective constants describing the rate of H₂O₂ degradation (k, s^–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H₂O₂. Catalases from variants 5 and 3 with high and low affinities for H₂O₂, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H₂O₂ can be used to obtain fungal variants producing extracellular catalases with improved properties.     

Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

Catalase (CAT_pp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K _m and k _cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k _in of the enzyme inactivation rate during the catalysis and the constant k ₂ of the interaction rate of the Complex I catalases with H₂O₂. Thermal inactivation of CAT_pp in solutions at 45°C was characterized by the effective rate constant k _in ^*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the firstorder rate constant k _in (US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k _cat/K _m, thermal stability comparable with the thermal stability of CAT in terms of k _in ^*, the minimal k _in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².     

Ultrasonic and Thermal Inactivation of Catalases from Bovine Liver,the Methylotrophic Yeast Pichia pastoris,and the Fungus Penicillium piceum

The kinetics of inactivation of catalases from bovine liver (CAT), the fungus Penicillium piceum (CAT1), and the methylotrophic yeast Pichia pastoris (CAT2) was studied in phosphate buffer (pH 5.5 or 7.4) at 45 and 50°C or under the conditions of exposure to low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm²). The processes were characterized by effective first-order rate constants (s^?1): k _in (total inactivation), k ^*_in (thermal inactivation), and k ^*_in (us) (ultrasonic inactivation). The values of k _in and k ^*_in increased in the following order: CAT1 < CAT < CAT2. Circular dichroic spectra of the enzyme solutions were recorded in the course of inactivation by high temperatures (45 and 50°C ) and LFUS, and the contents of secondary structures were calculated. Processes of thermal and ultrasonic inactivation of catalases were associated with a decrease in the content of α helices and an increase in that of antiparallel β structures and irregular regions (CAT1 < CAT < CAT2). We conclude that the enzymes exhibit the following rank order of resistance: CAT1 > CAT > CAT2. Judging from the characteristics of CAT1, it appears to be an optimum component for antioxidant enzyme complexes.

     

Use of cadmium hydroxide gel for isolation of extracellular catalases from Penicillium piceum and characterization of purified enzymes

We optimized the conditions for isolation of extracellular catalases from Penicillium piceum F-648 and P. piceum A3 by means of volume chromatography with cadmium hydroxide gel. Our study showed that 55–57 mg wet gel are sufficient for the maximum sorption of catalase from 1 ml of culture fluid. This gel was formed in 1 ml 70 mM Cd(NO₃)₂ after addition of NaOH (Cd(NO₃)₂/NaOH molar ratio 1: 2.2). The eluting solution contained 50 mM NaH₂PO₄(pH 7.0), 5.0 mM dithiothreitol, and 0.3% sodium cholate and was potent in desorbing catalase from the gel. Subsequent ultrafiltration of the eluate on the membrane with a retention limit of 50 kDa allowed us to concentrate and purify the sample from low-molecular-weight protein impurities. NH₄Cl (1.0 M) containing 0.3% sodium cholate was used to wash the sample from low-molecular-weight aromatic metabolites. Purified catalases included 33–34% antiparallel β-structures and 9%-spirals. Under optimal conditions in the medium of 10 mM phosphate buffered saline (pH 7.0) at 30°C, catalases from P. piceum F-648 were characterized by the following parameters: K _M, 158.8 mM; catalytic constant, 2.83 × 10⁶ s^?1; enzyme inactivation rate constant in H₂O₂ decomposition, 3.5 × 10^?2 s^?1; and constant of the interaction between catalase complex I and second molecule of H₂O₂, 1.8 × 10⁷M^?1 s^?1.     

Kinetics of Catalase Inactivation Induced by Ultrasonic Cavitation

Kinetic patterns of sonication-induced inactivation of bovine liver catalase (CAT) were studied in buffer solutions (pH 4.0–11.0) within the temperature range from 36 to 55^o. Solutions of CAT were exposed to LF (20.8 kHz) ultrasound (specific power, 48–62 W/cm²). The kinetics of CAT inactivation was characterized by effective first-order rate constants (s^–1) of total inactivation (k _in), thermal inactivation (*k _in), and ultrasonic inactivation (k _in(us)). In all cases, the following inequality was valid: k _in > *k _in. The value of k _in(us) increased with the ultrasound power (range, 48–62 W/cm²) and exhibited a strong dependence on the pH of the medium. On increasing initial concentration of CAT (0.4–4.0 nM), k _in(us) decreased. The three rate constants were minimum within the range pH 6.5–8.0; their values increased considerably at pH < 6.0 and pH > 9.0. At 36–55^o, the temperature dependence of k _in(us) was characterized by an activation energy (E _act) of 19.7 kcal/mol, whereas the value of E _act for CAT thermoinactivation was equal to 44.2 kcal/mol. Bovine and human serum albumins (BSA and HSA, respectively) inhibited sonication-induced CAT inactivation; complete prevention was observed at concentrations above 2.5 g/ml. Dimethyl formamide (DMFA), a scavenger of hydroxyl radicals (O ), prevented sonication-induced CAT inactivation at 10% (k _in and *k _in increased with the content of DMFA at concentrations in excess of 3%). The results obtained indicate that free radicals generated in the field of ultrasonic cavitation play a decisive role in the inactivation of CAT, which takes place when its solutions are exposed to low-frequency ultrasound. However, the efficiency of CAT inactivation by the radicals is determined by (1) the degree of association between the enzyme molecules in the reaction medium and (2) the composition thereof.     

Resistance of Penicillium piceum F-648 to Hydrogen Peroxide under Short-Term and Prolonged Oxidative Stress

Resistance of Penicillium piceumF-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H₂O₂ depends on the H₂O₂ concentration, time of exposure, and growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H₂O₂. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. piceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and a concentration of substrate less than 30 mM at pH more than 7.     

Inactivation of Glucose-6-Phosphate Dehydrogenase in Solution by Low- and High-Frequency Ultrasound

We compared the kinetics of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) inactivation in 0.1 M phosphate buffer (pH 7.4) at 36–50° under conditions of exposure to low-frequency (LF, 27 kHz, 60 W/cm²) or high-frequency (HF, 880 kHz, 1.0 W/cm²) ultrasound (USD). The inactivation of G6PDH was characterized by effective first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(usd), ultrasonic inactivation. Dilution of the enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k _in > k _in ^*. The rate constants increased with temperature. The Arrhenius plots of the temperature dependences of k _in and k _in(usd) had an break point at 44°C. The activation energy ( _act) of the total inactivation of G6PDH was higher than _act for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: _act was higher in the case of inactivation with low-frequency ultrasound (LF-USD) than high-frequency ultrasound (HF-USD). The rate of the ultrasonic inactivation of this enzyme substantially decreased in the presence of low concentrations of HO^. radical scavengers (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in solutions exposed to USD.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Stability of Glucose 6-Phosphate Dehydrogenase Complexed with Its Substrate or Cofactor in Aqueous and Micellar Environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), or cofactor, NADP⁺, has been studied within the range 20–40°C in three media: (a) 0.04 M NaOH–glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, k _in(s^–1). In the case of G6PDH–NADP⁺complexes, the values of k _inwere independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of k _infor the complex G6PDH–GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of k _incorresponded to the degree of hydration W ₀= 16.7; at W ₀> 16.7 and W ₀< 16.7, k _inincreased. Within the range 20–40°C, the values of k _inmeasured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of k _inwere characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6°C to 40°C. In all media studied, NADP⁺complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH–NADP⁺melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

Diversity and enzyme properties of protease-producing bacteria isolated from sub-Antarctic sediments of Isla de Los Estados, Argentina

Protease-producing bacteria isolated from sub-Antarctic marine sediments of Isla de Los Estados (Argentina) were characterized, and the thermal inactivation kinetics of their extracellular proteases compared. Isolates were affiliated with the genera Pseudoalteromonas, Shewanella, Colwellia, Planococcus, and a strain to the family Flavobacteriaceae. Colwellia strains were moderate psychrophiles (optimal growth at about 15°C, maximum growth temperature at around 25°C). 16S rRNA phylogenetic analysis revealed that these strains and Colwellia aestuarii form a distinct lineage within the genus. The remaining isolates were psychrotolerant and grew optimally between 20 and 25°C; two of them represent potentially novel species or genus (16S rRNA < 97% sequence similarity). The thermostability of the extracellular proteases produced by the isolates was analysed, and the inactivation rate constant (k _in), the activation energy (Ea_in) and the activation Gibbs free energy of thermal inactivation (ΔG ^* _in) determined. ΔG ^* _in, calculated at 30°C, varied between 97 and 124 kJ/mol. Colwellia enzyme extracts presented the highest thermosensitivity, while the most thermostable protease activity was shown by Shewanella spp. These results demonstrated that the stability to temperature of these enzymes varies considerably among the isolates, suggesting important variations in the thermal properties of the proteases that can coexist in this environment.     

Cloning and Characterization of Catalases from Rice,Oryza sativa L.

Catalase is the major H₂O₂-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)₆-tagged proteins in Escherichia coli. The recombinant (His)₆-polypeptides were enriched to apparent homogeneity and characterized. With H₂O₂ as substrate, the highest catalase k _cat value (20±1.71×10^?3 min^?1) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.     

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm² were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0°C and characterized by effective first-order rate constants of US inactivation k _in (us) in min^–1. Values of k _in (us) depend on the specific ultrasonic power within the range of 20-60 W/cm², on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO^· radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0°C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm² on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.     

Flavonoids: Efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44°C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm²). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k _in, k*_in, and k _in(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01–50 μM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol ? astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO^· and O ₂ ^·? in G6PDH inactivation in solutions.     

Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP⁺ was characterized in aqueous solutions at 36–47°C under treatment with low frequency (27 kHz, 60 W/cm²) and high frequency ultrasound (880 kHz, 1 W/cm²). To this end, we measured three effective first-order inactivation rate constants: thermal k _in ^* , total (thermal and ultrasonic) k _in, and ultrasonic k _in(US). The values of the constants were found to be higher for the free enzyme than for its complexes GPDH-GP and GPDH-NADP⁺ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (E _a) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (ΔH*, ΔS*, and ΔG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of E _a and ΔH* values in comparison with the corresponding values for thermal inactivation. The E _a, ΔH*, and ΔS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the ΔH* and ΔS* values was observed for all the inactivation processes for free GPDH and its complexes.     

An ultrasonic inactivation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> glucose oxidase in aqueous solutions

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37–59°C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constantsk _in, k*_in andk _in(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO^·—DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Precipitated,Coprecipitated, and Carbon–Mineral Sorbents for Isolation of Exracellular Catalase from Penicillium piceum F-648

The abilities of various sorbents to adsorb catalase (CAT; EC 1.11.1.6) from filtered culture liquid (FCL) of the fungus Penicillium piceum F-648 were compared. Potassium phosphate, hydroxyapatite (HAP), and coprecipitated sorbents containing calcium phosphate and magnesium hydroxide adsorbed extracellular CAT more efficiently than aluminum oxide, aluminum phosphate, or quartz sand. The enzyme was isolated from FCL of Penicillium piceum with the use of HAP and a binary coprecipitated sorbent, Ca₃(PO₄)₂ + Mg(OH)₂, 1 : 1 (CM). The CAT(CM) sample contained the least amount of protein admixture. Its spectra had absorption maximums at 279.6, 406.8 (Soret band), 540, 585, 636, and 703 nm and negative molar ellipticity minimums at 207 and 210–214 nm. The kinetic indices of the samples (K _M, V _max : K _M, and specific activity) were intricately dependent on the protein concentration in the reaction mixture. In dilute solutions, the K _M and specific activities of CAT(CM) and CAT(HAP) equaled 667 and 137 mM; 300.9 × 10⁴ and 30.0 × 10⁴ U/mg protein, respectively. The effective velocity constants of inactivation of CAT(HAP), CAT(CM), and FCL in the reaction of H₂O₂ decomposition increased dramatically after the dilution of samples. In the infinitely dilute solution, they were 4.30 × 10^–2, 6.46 × 10^–2, and 1.12 × 10^–2 s^–1, respectively.     

On the turnover and proteolysis of catalase in tissues of the guinea pig and acatalasemic mice.

Turnover characteristics (half-lives and rate constants for synthesis and degradation) have been determined for the catalases of guinea pig and three different strains of mice by means of the kinetics of return of enzyme activity after inhibition with 3-amino-1,2,4-triazole. The catalase of hypocatalasemic mice (strain C_sD) did not display an appreciably different half-life to that of the wild-type mice, but catalase in the tissues of acatalasemic mice (strain C_sB) exhibited a half-life which was only half that of the wild type, while the half-life of guinea pig catalase was more than twice that of wild-type mice. Significant differences were also noticed in regard to the in vitro susceptibility of the catalases of these animals to protease inactivation. Large-granule (lysosomal, mitochondrial and peroxisomal) extracts proved far more susceptible to protease inactivation than cytosol extracts, and marked changes in the heteromorph pattern of mouse liver cytosol catalase were observed to accompany limited proteolysis. These results support the conclusion that the in vitro susceptibility of proteases may be an important determining factor in the rate of degradation of an enzyme in vivo.