John L. Mego

Further evidence for a proton pump in mouse kidney phagolysosomes: effect of nigericin and 2,4-dinitrophenol on the stimulation of intralysosomal proteolysis by ATP.

Abstract The lipid soluble acid 2,4-dinitrophenol completely abolished the stimulatory effect of ATP on intralysosomal proteolysis in mouse kidney phagolysosomes at pH 8. The protonophore had no effect in the absence of ATP at pH 8 but inhibited intralysosomal proteolysis in unbuffered media. The ionophorous antibiotic nigericin also prevented the action of ATP at pH 8 but had no effect in the absence of ATP in unbuffered media. Nigericin also inhibited intralysosomal proteolysis at pH 8 in the absence of ATP. These observations support the hypothesis that the phagolysosome membrane contains an ATP-driven proton pump which functions to maintain intralysosomal acidity.

An effect of cadmium on heterolysosome formation and function in mice

Abstract The effects of cadmium acetate injections on heterolysosome formation and on the digestion of 121I-labeled formaldehyde-treated bovine albumin in these particles from the kidneys and livers of mice have been studied. Cadmium inhibited proteolysis in liver particles 2 hr after intraperitoneal injections at LD 50 doses (4.3 mg Cd/kg), and the effect became progressively more pronounced up to 20 hr. At this time, kidney particles were also affected but to a lesser degree. Mice surviving the cadmium injections recovered their capacity to degrade intralysosomal protein. The most pronounced effect of cadmium was on the uptake of labeled protein into subcellular particles, particularly in the liver at 24 hr after injection. Cadmium ion also caused the disruption of subcellular osmotically active particles (heterolysosomes) containing injected protein when added to particulate suspensions centrifuged from kidney and liver homogenates. Injections of cadmium acetate inhibited proteolysis in isolated liver heterolysosomes prepared 1 hr after intravenous injections of a relatively large dose of nonradioactive formaldehyde-treated albumin. This suggested that the metal prevented recovery of the capacity for normal intralysosomal proteolysis, perhaps by interfering with primary lysosome formation.

Stimulation of intralysosomal proteolysis by cysteinyl-glycine, a product of the action of γ-glutamyl transpeptidase on glutathione

The mechanism of the stimulatory effect of glutathione on proteolysis in mouse kidney lysosomes and a lack of an effect in lysosomes from the liver was investigated. The stimulation in kidney lysosomes was inhibited by serine plus borate, a reversibly inhibitor of gamma-glutamyl transpeptidase. Treatment of mouse kidney lysosome suspensions with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), an irreversibly inhibitor of the transpeptidase, also inhibited the effect of glutathione, but this inhibition was completely relieved by washing and addition of freshly prepared kidney membranes or purified gamma-glutamyl transpeptidase to the incubation mixtures. Cysteinyl-glycine, a product of the action of gamma-glutamyl transpeptidase, stimulated proteolysis in acivicin-inhibited kidney lysosome preparations similarly to glutathione, and cysteine had no effect at equivalent concentrations. Glutathione also stimulated proteolysis in liver lysosomes in the presence of washed kidney membranes or gamma-glutamyl transpeptidase, but the effect was similar to that produced by equivalent concentrations of cysteine. These results suggest that the stimulatory effect of glutathione was mediated by the action of gamma-glutamyl transpeptidase present in contaminating cell membrane fragments in the lysosome preparations, and that glutathione does not take part in intralysosomal proteolysis. However, the possibility that cysteinyl-glycine is a physiological intralysosomal disulfide reductant in kidney lysosomes has not been excluded.

Lysosomal membrane adenosine triphosphatase; solubilization and partial characterization

Abstract Membranes prepared from Triton WR-1339-filled lysosomes (tritosomes) contained ATPase activity with a pH optimum of 5–8. These membranes also showed adenosine diphosphatase, adenosine monophosphatase, acid β-glycerol phosphatase, and acid pyrophosphatase activities. The soluble (nonmembrane) fraction of the tritosomes also contained these activities, but the properties of the soluble adenine nucleotide phosphatase activities were different from the membrane-associated enzymes. The pH optimum of tritosomal membrane ATPase changed to 5 after solubilization with Triton X-100, but ADPase and AMPase optima remained at 6–7. The pH optimum of intact membrane ATPase was also 5 when the substrate was α,β-methylene-ATP. Thus, tritosomal membrane ATPase apparently exhibits a pH 8 optimum only when acting in concert with ADPase and AMPase in intact membranes. Rates of ATP hydrolysis to adenosine were also significantly greater in intact membranes than in Triton X-100-solubilized fraction. Centrifugation of Triton X-100-solubilized tritosomal membranes in sucrose density gradients showed that ATPase and ADPase activities sedimented to one peak, and that AMPase, acid phosphatase, and pyrophosphatase were grouped in another peak. Thus, tritosomal membrane ATPase activity was not due to the latter enzymes. The resulting purification was about fourfold for ATPase. The Mr for ATPase and ADPase was estimated to be about 65,000 and for AMPase, acid phosphatase, and pyrophosphatase about 200,000.

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization.

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.

Inhibition of heterolysosome formation and function in mouse kidneys by injection of mercuric chloride.

Abstract Intravenous or intraperitoneal injections of mercuric chloride (5 mg Hg/kg) into mice resulted in an inhibition of proteolytic activity in isolated kidney heterolysosomes containing intravenously injected denatured (formaldehyde) 125 I-albumin but not in these particles from the liver. The inhibition occurred if the mercuric chloride was injected up to about 17 hr before injection of labeled protein. The proportion of particle-bound radioactivity which could be released by osmotic shock was substantially decreased in subcellular suspensions from mercury-injected mice. In some experiments therefore, digestive rates were normal in mercury-injected mice if proteolysis was measured in terms of the osmotically releasable radioactivity rather than in terms of the total particle-bound material. This suggest that mercury inhibited either endocytosis of labeled protein or heterolysosome formation. However, in most experiments in which digestion was measured in the presence and absence of mercaptoethanol, stimulation of proteolysis by mercaptoethanol was significantly greater in heterolysosomes from mercury-injected animals than in the controls. This suggests that mercury affected proteolytic activity within the organelles as well as heterolysosome formation.

Effects of some antimalarials and related substances on intralysosomal proteolysis

Abstract The antimalarials primaquine, chloroquine, quinine, and quinacrine and the related substances 6-chloro-9-(3′-dimethylamino-2′-hydroxypropylamino)-2-methoxyacridine and 9-amino acridine inhibited intralysosomal proteolysis at pH 8 in mouse kidney subcellular suspensions containing [ 125 I]-labeled albumin-filled phagolysosomes at concentrations from 5 × 10 −5 (quinacrine) to about 2.5 × 10 −4 M (quinine). All these substances inhibited rat liver cathepsin B 1 but had no effects on cathepsins A, C or D. These inhibitions were greater on the alkaline side of the pH optimum of cathepsin B 1 . The drugs also partially inhibited the stimulatory effect of ATP on intralysosomal proteolysis at pH 8 which we have interpreted previously to reflect an energy-dependent proton pump for maintenance of intralysosomal acidity. Preincubation of [ 125I ]-labeled albumin-filled phagolysosomes for 10 min at pH 8 in media containing 0.1 mM quinacrine or chloroquine or 0.5 mM quinine sulfate, followed by washing to remove these drugs, partially inhibited the stimulatory effect of ATP on further incubation in antimalarial-free media but had no effects on intralysosmal proteolysis. Preincubation in 1 mM chloroquine completely abolished the stimulatory effect of ATP but also partially inhibited proteolysis. Antimalarials also inhibited rat liver lysosomal (tritosomal) membrane ATPase suggesting that this activity may be related to the stimulatory effects of ATP on intralysosomal proteolysis. Quinine was the most effective of the inhibitors tested. These studies suggest that [ 125 I]-labeled albumin-filled mouse kidney phagolysosomes may provide a convenient model system for rapid screening of drugs potentially useful for the treatment of diseases caused by organisms possessing a lysosome-like digestive system.

Effect of mycotoxins on uptake and degradation of [125I] albumin in mouse liver and kidney lysosomes.

Some mycotoxins have been evaluated with respect to effects on the reticuloendothelial function of uptake and degradation of a soluble, denatured protein in phagolysosomes isolated from mouse liver and kidneys. Toxins were dissolved in dimethylsulfoxide and administered intraperitoneally. Particulate fractions isolated from liver and kidney homogenates were assayed for proteolytic activity within osmotically active particles (phagolysosomes). Treatment with citrinin, penitrem A, streigmatocystin, and zearalenone produced inhibition of proteolysis in kidney but not in liver phagolysosomes. Moniliformin and T-2 toxin had no detectable effect on lysosome formation or function in either tissue. The phagolysosomal toxicity of these compounds appeared to be more toward kidney than liver. Both sterigmatocystin and zearalenone caused an increase in kidney phagolysosomal fragility at early times after treatment, and all agents except penitrem A caused a decrease in uptake of labeled albumin into kidney cells. Citrinin labelized kidney phagolysosomes in vitro but not in vivo, while only citrinin and zearalenone inhibited in vitro preparations of cathepsin.

Alcohol dehydrogenases of Euglena gracilis, strain Z.

Abstract Cell-free extracts from heterotrophically grown Euglena gracilis were assayed for alcohol dehydrogenase activities by the reduction of NAD with various alcohols as substrates. Highest rates were obtained with isopropanol and cinnamyl alcohol. Isopropanol was oxidized at about twice the rate of ethanol. Mercaptoethanol inhibited isopropanol dehydrogenase activity but had no effect on ethanol oxidation.

The effect of carbon tetrachloride on lysosome function in kidneys and livers of mice

Abstract Lysosomal function in the degradation of intravenously injected 125 I-labelled albumin was evaluated in carbon tetrachloride-injected and control mice. A transient effect was noted in the kidneys at 1 h after injection of the solvent, but at later times the effects on phagolysosome formation and function were minimal and were similar in liver and kidneys. The results suggest that lysosomes are not involved in the hepatotoxicity of carbon tetrachloride.