Isolation of a cardiotonic glycoprotein,striatoxin, from the venom of the marine snail Conus striatus

Striatoxin, a powerful cardiotonic glycoprotein has been isolated from the venom of the marine snail Conus striatus, monitored by the inotropic action on the guinea-pig left atria. The molecular weight was estimated to be 25,000 by gel filtration. The purified glycoprotein is electrophoretically homogeneous. The toxin at concentrations above 10^?7 g/ml has a long-lasting inotropic action, which was abolished by tetrodotoxin (10^?6 M). The minimum lethal dose in the fish Rhodeus ocellatus smithi was 1 μg/g body weight. A possible biological role of striatoxin in C. striatus is briefly discussed.     

A novel response to propranolol: Contractile response in the isolated rabbit ear artery

Propranolol caused a contractile response in the isolated rabbit ear artery (EA). The concentration of propranolol causing a threshold contraction was 1.76 × 10^?6M while that causing a maximal contraction of 2.2 ± 0.18 g was 3 × 10^?5M. Higher concentrations caused tissue relaxation. Phentolamine, 10^?7 and 10^?6M reduced the propranolol-induced contractions by 50% and 90%, respectively while prazosin, 10^?8, 10^?7 and 10^?6M caused reductions of 54, 74 and 88%, respectively. Reserpinization of the rabbit with 5 mg/kg 24 hours before use eliminated the EA contractile response to tyramine but had no effect on that to propranolol. Desmethylimipramine plus deoxycorticosterone acetate enhanced the submaximal contraction of the EA to propranolol. $\text{In vitro}$ denervation with 6-hydroxydopamine (6-OHDA) decreased the response of the EA to tyramine and propranolol by 96% and 85% respectively but increased that to norepinephrine (NE) by 11%. Rabbit thoracic aorta (TA) did not respond to propranolol. In EA contracted with vasopressin o or 30 mM potassium, propranolol 10^?4 and 3 × 10^?4M caused a 20% and 100% relaxation, respectively. It is concluded that propranolol elicits a contractile response in the EA, at least in part, by direct activation of postsynaptic alpha adrenoceptors.     

The effect of SDS on protein zone dispersion in polyacrylamide gel electrophoresis

The zone dispersions of the reduced subunit of β-lactoglobulin B and its derivative with sodium dodecyl sulfate (SDS) were measured during polyacrylamide gel electrophoresis (PAGE) using the apparatus for continuous optical scanning at 280 nm. The ratio of apparent diffusion coefficients (D′) of the reduced subunit of β-lactoglobulin B (1.71 × 10^?6 cm²/s) and of its SDS-derivative (7.1 × 10^?7 cm²/s) was found to be 2.4 under the conditions of PAGE (pH 10.4, 0.015 ionic strength, 1°C, 4 mA/cm² current density, 50 μg protein load, 10% T gel) used. This is nearly twice the value of 1.3 predicted, under the assumption of sphericity for these protein molecules, on the basis of the binding of 1.4 g of SDS per gram of protein. It is postulated that the increment in zone sharpness (decrease in apparent diffusion coefficient) over that predicted by SDS binding alone is a general property of SDS-proteins providing gel electrophoresis in SDS-containing buffers with a resolving power larger than that obtained in the absence of the detergent.     

Purification of rat chondrosarcoma xylosyltransferase

The chondroitin sulfate chain-initiating enzyme, UDP-d-xylose:core protein β-d-xylosyltransferase has been purified over 600-fold from the high-speed supernatant fraction of a rat chondrosarcoma. The purification procedure involved differential centrifugation, gel chromatography on Sephadex G-200, and affinity chromatography on a matrix consisting of core protein bound to Sepharose. The purified enzyme was homogenous by electrophoretic and immunological criteria, had a molecular weight between 95,000 and 100,000 and contained approximately 10% carbohydrate. The K_m value for UDP-xylose was 1 × 10^?5, m and for the core-protein acceptor was 330 mg/liter.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

NADP-malic enzyme from maize leaf: purification and properties.

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C⁴ pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s_20,w) of 9.7 and molecular weight of 2.27 × 10⁵ in sucrose density gradient centrifugation, and molecular weight of 2.26 × 10⁵ calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO₃^? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K_m (malate), 0.4 mm; K_m (NADP), 17.6 μm; K_m (Mg²⁺), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol.     

Self-association in highly concentrated solutions of myoglobin: a novel analysis of sedimentation equilibrium of highly nonideal solutions

The sedimentation equilibrium in concentrated solutions of hemoglobin and myoglobin has been measured. The ratio of the apparent molecular weight of hemoglobin to that of myoglobin. R. is found to obey the empirical relation R(c) = 3.8–4.25 × 10^?2c+6.44 × 10^?4c² ? 2.21 × 10^?5c³, where c is the protein concentration in g/dl. for c??40 g/dl. A theoretical relation for the dependence of R upon c in the absence of protein self-association is presented. This relation cannot be fitted to the experimental results, and the discrepancy is attributed to self-association of myoglobin.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 ± 2800 in Tris · HCl buffer and 21 000 ± 1 000 in 6 M guanidine · HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10^?3 M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 · 10⁶ M^?1 for the apparent association constant and 4.4 · 10^?4 mol of Ca²⁺ bound per g of protein for high affinity binding sites were estimated, on the basis of data from the equilibrium dialysis. The origin possible biological role of this protein is discussed.     

Fate of naturally occurring epoxy acids: a soluble epoxide hydrase, which catalyzes cis hydration, from Fusarium solani pisi.

A cell-free extract prepared from Fusarium solani pisi grown on cutin, catalyzed the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid to 9,10,18-trihydroxyoctadecanoic acid while extracts from glucose-grown cells contained <6% of this activity. The product was identified by Chromatographic techniques and by radio gas-liquid chromatography of its periodate oxidation products. This epoxide hydrase activity had a pH optimum at 9.0 and it was located mainly in the 100,000g supernatant fraction. Rate of hydration of the epoxy acid was linear up to 15 min and up to a protein concentration of 30 μg/ml. This fungal epoxide hydrase has a molecular weight of 35,000, as determined by Sephadex G-100 gel filtration. It was partially purified by ammonium sulfate fractionation and gel filtration. The apparent K_m and V of the enzyme was 2 × 10^?4m and 222 nmoles/min/mg, respectively. Parachloromercuribenzoate strongly inhibited the enzyme, while N-ethylmaleimide was a less potent inhibitor. 1,1,1,-Trichloropropylene-2,3-oxide at 10^?3m gave 50% inhibition of the hydration of 18-hydroxy-9,10-epoxyoctadecanoic acid. Kinetic analysis showed that trichloropropylene oxide was a competitive inhibitor. 18-Acetoxy-9,10-epox-yoctadecanoic acid, methyl 18-acetoxy-9,10-epoxyoctadecanoate, 9,10-epoxyoctadecanoic acid, and styrene oxide were not readily hydrated by this fungal epoxide hydrase showing that it has a stringent substrate specificity. Analysis of the enzymatic hydration product on boric acid-impregnated silica gel plates showed that the product obtained from the cis epoxide was exclusively erythro while acid hydrolysis of this epoxide gave rise to the expected threo product. This enzyme is novel in that it catalyzes cis hydration of epoxide while the other epoxide hydrases heretofore isolated catalyzed trans hydration of epoxides.     

Purification of the chloroplastic valyl-tRNA synthetase from Euglena gracilis.

Euglena gracilis chloroplast valyl-tRNA synthetase was purified 990 fold to a specific activity of about 1100 units/mg protein, by a series of steps including ammonium sulfate precipitation and chromatography on hydroxyapatite, DEAE-cellulose, Blue Dextran — Sepharose and Sephadex G200. The enzyme gives a single band upon polyacrylamide gel electrophoresis, appears to be a monomer with a molecular weight of 126,000 daltons and has Km values of 1.5 × 10^?5 M for L-valine, 5 × 10^?5 M for ATP, and 6 × 10^?8 for tRNA^Val.     

Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

The extent of increase in the activity of phenylalanine ammonia-lyase upon illumination for 15 hr of cultured Petroselinum hortense cells was greatly dependent upon the age of the culture. Two distinct peaks in specific activity were observed during the growth cycle, one occurring at the beginning and the other at the end of the period of increase in cell fresh weight. High yields in both cell fresh weight and enzyme activity were obtained with the second peak shortly before the stationary phase of the culture was reached. Cells were harvested at this stage and stored at ?20 °C.The enzyme was purified from the frozen cells to apparent homogeneity by precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose, Sephadex G-200 and hydroxyapatite columns, and preparative polyacrylamide gel electrophoresis. An over-all yield of 16% was achieved with a 440-fold increase in the specific activity by purification of the enzyme through the hydroxyapatite step.Upon analytical polyacrylamide gel electrophoresis either in the presence or in the absence of sodium dodecyl sulfate, the purified protein migrated essentially as a single band. Molecular weights of about 330,000 and 83,000, respectively, were estimated for the enzyme and for its protein subunits. Thus, the enzyme molecule seems to be composed of four probably identical protein subunits. Two Michaelis constants for l-phenylalanine (K_m^L, 3.2 × 10^?5 M, and K_m^H, 2.4 × 10^?4 M) and a Hill coefficient of h = 0.6 were obtained. This suggests that the enzyme is subject to regulation of its catalytic properties by negative cooperativity of the protein subunits.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 ± 20 mM^?1 · cm^?1. The molecular weight based on protein and chromophore assays was found to be 1.5 · 10⁵; the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.     

Purification and characterization of selenium-glutathione peroxidase from hamster liver

Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 μmol cumene hydroperoxide reduced/min/mg of protein at 37 °C, pH 7.6, and 0.25 mm GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 °C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k₁ (t-butyl hydroperoxide), 7.06 × 10⁵ mm min^?1; k₁ (cumene hydroperoxide), 1.04 × 10⁶ mm^?1 min^?1; k₁ (p-menthane hydroperoxide), 1.2 × 10⁶ mm^?1 min^?1; k₁ (diisopropylbenzene hydroperoxide), 1.7 × 10⁶ mm^?1 min^?1; k₁ (linoleic acid hydroperoxide), 2.36 × 10⁶ mm^?1 min^?1; k₁ (ethyl hydroperoxide), 2.5 × 10⁶ mm^?1 min^?1; and k₁ (hydrogen peroxide), 2.98 × 10⁶ mm^?1 min^?1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.