H+ stoichiometry of sites 1 + 2 of the respiratory chain of normal and tumor mitochondria

The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K+ (+ valinomycin) and Ca2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and tumor mitochondria with beta-hydroxybutyrate and other NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.     

Membrane (Na+ + K+)-ATPase of canine brain,heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures

Effects of commonly used purification procedures on the yield and specific activity of (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, Na⁺ + K⁺-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na⁺ + K⁺)-ATPase and the ratio between (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na⁺ + K⁺)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na⁺ + K⁺)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.     

Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+

The activity of phosphodiesterase (“Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca²⁺ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca²⁺. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca²⁺ binds to the modulator, could explain the stimulatory effect of Ca²⁺ on this enzyme as follows: The “Ca²⁺ plus Mg²⁺-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca²⁺) complex, and Ca²⁺, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca²⁺) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca²⁺, the process is reversible.     

The effect of gentamicin on calcium uptake by renal mitochondria

The effect of the nephrotoxic aminoglycoside antibiotic, gentamicin, on calcium uptake by renal cortical mitochondria was assessed in vitro. Gentamicin was found to be a competitive inhibitor of mitochondrial Ca⁺⁺ uptake. This effect displayed a dose response with a K_i of 233 μM and occurred at gentamicin concentrations below those that inhibit mitochondrial electron transport. These results further demonstrate the potential for gentamicin to alter membrane function and thereby contribute to toxic cell injury via its interactions with divalent cations.     

Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase.

The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate ³²P from [γ-³²P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca²⁺ ATPase and Mg²⁺ ATPase activities without any changes in Na⁺K⁺ ATPase activity. The observed increase in $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity was found to be associated with an increase in V_max value of the reaction whereas K_a value for $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.     

The phosphorylation potentials generated by respiring Ehrlich ascites tumor mitochondria

The extra- and intramitochondrial phosphorylation potentials (ΔG_p(out) and ΔG_p(in), respectively) generated by respiring Ehrlich ascites tumor mitochondria were determined, using succinate, pyruvate + malate, ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, and ascorbate + ferrocyanide as substrate systems. Values of ΔG_p(out) exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) in post-ADP state 4 respiration were found with succinate as substrate, in agreement with data on normal rat liver mitochondria. ΔG_p(out) values exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) were also observed with ascorbate + TMPD or ascorbate + ferrocyanide as substrates. Slightly lower values of ΔG_p(out) were found with the NAD-linked substrates pyruvate + malate. The intramitochondrial ΔG_p(in) developed by respiring Ehrlich ascites tumor mitochondria respiring on succinate approached 12 kcal mol^?1 (50.2 kJ mol^?1), in agreement with reported values on rat liver mitochondria. The prior accumulation of Ca²⁺ and phosphate by the Ehrlich cell mitochondria did not lower the extramitochondrial ΔG_p(out) developed after a subsequent addition of ADP. Although the rate of oxidative phosphorylation of Ehrlich ascites tumor cells is reduced by intramitochondrial Ca²⁺ and phosphate (Villalobo and Lehninger (1980) J. Biol. Chem., 255, 2457–2464) they are still capable of generating ATP in the suspending medium against a high thermodynamic gradient, as expressed by the [ATP]/[ADP][P_i]mass action ratio.     

Effect of extracellular Ca2+ on the morphogenesis of Dictyostelium discoideum.

Effect of extracellular Ca²⁺ on the morphogenesis of the cellular slime mold Dictyostelium discoideum was examined on agar plate. The concentration of Ca²⁺ in agar plate was controlled by keeping the concentration of a chelating reagent EGTA constant and varying the concentration of total calcium. From experiments in which EGTA concentration was kept at 2.0 × 10^?3 M, it was found that by decreasing Ca²⁺ concentration the morphogenesis was modified so that development of the aggregating amebae into fruiting bodies was accelerated and the period of migrating slugs was shortened. Below 1.0 × 10^?3 M of Ca²⁺ concentration, the total number of aggregates initially increased with decreasing Ca²⁺ concentration, reached a maximum at about 3.0 × 10^?7 M of Ca²⁺ concentration and hereafter decreased with decreasing Ca²⁺ concentration. The number of mature fruiting bodies obtained at 36 h period after starvation depends on Ca²⁺ concentration and the total number of aggregates. The cell aggregation initiated at the same time period after starvation even at an extreme case of 1.0 × 10^?8 M of Ca²⁺ concentration as under enough Ca²⁺ supply, while the formation of mature fruiting body was seriously inhibited. These observation suggested that the cAMP-mediated cell aggregation in D. discoideum is a Ca²⁺-independent phenomena, although extracellular Ca²⁺ is necessary for the normal development of the aggregated amebae.     

Sperm binding to eggs of Ciona intestinalis. Role of Ca2+

In this paper we show that in the ascidia Ciona intestinalis extracellular Ca2+ is required for the binding of the spermatozoa to the vitelline coat (VC) glycerol-treated eggs and for fertilization to occur. Divalent cations, Mg2+ and Mn2+, cannot replace Ca2+. Once bound, the spermatozoa cannot be detached from the vitelline coat by adding of EGTA. Verapamil does not interfere with the binding of spermatozoa to the vitelline coat, whereas it blocks the Ca2+ ionophore A23187-induced sperm activation and acrosome reaction. Fertilization too was inhibited by the presence of this drug.     

Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography

Covalently closed circular DNA can be isolated rapidly from cell lysates in a two-step process. Hydroxylapatite chromatography to prepurify the plasmid DNA from contaminating protein and RNA is followed by a step gradient elution of covalently closed circular (CCC) plasmid DNA from an acridine yellow affinity column. This procedure results in CCC DNA of a purity comparable to that obtained from ethidium bromide-CsCl gradients without lengthy centrifugation and free of contaimination by intercalating dye. Up to 250 μg of CCC pBR 322 can be isolated from 500 ml of bacterial culture in 4–6 h.     

The effect of A13+ on the physical properties of membrane lipids in Thermoplasma acidophilum.

Using Electron Paramagnetic Resonance Spectroscopy, Al³⁺ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism $\text{Thermoplasma}\text{acidophilum}$ at a pH > 2. The ability of Al³⁺ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, 10^?2 M Al³⁺ increased the lower lipid phase transition by 39°C, and a detectable change was observed with AlCl₃ concentrations as low as 10^?5 M. The ability of Al³⁺ to increase the lower lipid phase transition temperature of $\text{T.}\text{acidophilum}$ is the largest of any cation/lipid interaction yet reported.     

Evidence for dysfunction in the regulation of cytosolic Ca2+ in excitation-contraction uncoupled dysgenic muscle

In noncontracting, dysgenic murine muscle, excitation is uncoupled from contraction. To test whether the gene lesion is expressed as a defect in the regulation of the intracellular free Ca2+ levels, cultured normal and dysgenic muscle at various stages of development (proliferative myoblasts, early, late, and mature myotubes) were exposed to increasing increments (0.5-mM steps) of extracellular Ca2+ in ionophore A23187-Ca2+-EGTA-buffered media. Normal and dysgenic muscle at all stages (except myoblast) displayed contractures at approximately 500 microM free Ca2+ and higher. Experiments using finer increments of Ca2+ and different ionophore concentrations indicated an external Ca2+ threshold for contracture at 265 microM Ca2+ for early and late myotubes and 47-78 microM for mature normal and dysgenic myotubes. Low extracellular concentrations of calcium (14 microM and 0.76 nM) caused elongation of both normal and dysgenic myotubes. Mature cells were depolarized by exposure to increasing extracellular K+ and monitored by intracellular recording; normal and dysgenic myotubes showed similar reductions in membrane potentials. Depolarization to -35 mV elicited contractures in normal myotubes, but even depolarization to -9 mV in dysgenic cells elicited no response. Thus steady-state depolarization of dysgenic muscle does not cause contractures, which can, however, be elicited by increasing the intracellular free Ca2+. These results offer new evidence for a possible defect in the regulation of Ca2+ levels in dysgenic muscle.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Histamine H2-receptors in the gastric mucosa: role in acid secretion.

Currently, two major hypotheses dominate thinking about the role of histamine in the regulation of gastric acid secretion. Code has proposed that histamine is the final common mediator of secretagogue action on the parietal cell while Konturek and Grossman have suggested a multi-receptor control of the secretory process. Experimental results derived from the use of recently synthesized histamine H₂-receptor antagonists have been used by both groups to support their hypotheses. Paradoxically, these hypotheses depend on the presumed specificity of the H₂-antagonists in blocking histamine mediated acid secretion while the apparent lack of such secretagogue specificity of the H₂-antagonists is an important basis for the development of the hypotheses. Our review will analyze the experimental evidence which implicates the histamine H₂-receptor in the control of hydrogen ion secretion as well as evidence for and against receptor specificity in the gastric mucosa of histamine H₂-receptor antagonists.     

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Partial characterization of the protein components of eosinophil granules isolated from guinea pig exudates

Peritoneal exudates enriched in eosinophils were induced in guinea pigs by serial intraperitoneal injections of Trichinella antigen. A method is described whereby highly purified eosinophil granules were obtained in good yield from these exudates. The granules were shown by electron microscopy to be intact and comparable to those of the mature eosinophil. The proteins of the purified granules were completely extracted with cetyltrimethylammonium bromide (CETAB) and were examined by polyacrylamide gel electrophoresis. Four major and several minor proteins, all of them basic, were resolved. One of the major proteins was identified as eosinophil peroxidase. Acid phosphatase, β-glucuronidase, and arylsulfatase activities were also present as minor components.