The Role of Atp in Lysosomal Cystine Efflux

Smith, Micah J.; Potashnik, R.; Greene, Alice A.; Jonas, Adam J.; Schneider, Jerry A.

doi:10.1203/00006450-198404001-00799

Cited by 7 publications

(9 citation statements)

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“…Since a focus of this study was to define the characteristics of ADA functioning within the environment of the lysosomal compartment, all assays were performed at pH 5.5, which is near the intralysosomal pH previously reported for human fibroblast lysosomes [29]. Lysosomal ADA activity was found to be directly dependent on the amount of lysosomal material added to incubation mixtures ( Figure 3), and assays were linear with time until a plateau was reached when all adenosine had been converted into inosine (results not shown).…”

Section: Resultsmentioning

confidence: 99%

Demonstration of adenosine deaminase activity in human fibroblast lysosomes

Lindley

Pisoni

1993

Biochemical Journal

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Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation.

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Section: Resultsmentioning

confidence: 99%

Demonstration of adenosine deaminase activity in human fibroblast lysosomes

Lindley

Pisoni

1993

Biochemical Journal

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“…The pH of lysosomes can be sensed by fluorescent dyes such as acridine orange [15-181 and fluorescein isothiocyanate (F1TC)-dextran [19][20][21][22]. Acridine orange is a weak base and the non-ionized, but not the ionized, form of this fluorescent dye diffuses across membranes readily.…”

Section: Methods Of Studying Lysosomal Phmentioning

confidence: 99%

Lysosomal pH: a link between cell volume and metabolism

Läng

Busch

Völkl

et al. 1994

Biochemical Society Transactions

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“…In transformed human lymphoblasts, cystine transport has been found to be dependent on ATP-stimulated lysosomal acidification (Jonas et al, 1982a,b). However, other studies using human leucocytes and fibroblasts have shown that cystine transport, although stimulated by Mg2+, occurs in the absence of ATP (Gahl et al, 1983(Gahl et al, , 1984aSmith et al, 1984). N-Ethylmaleimide and CCCP, which inhibit ATP-mediated lysosomal acidification, inhibit ATPstimulated cystine transport in human lymphoblasts, but have had no effect on transport in other studies (Gahl et al, 1983(Gahl et al, , 1984aJonas et al, 1982bJonas et al, , 1983.…”

Section: Introductionmentioning

confidence: 97%

“…N-Ethylmaleimide and CCCP, which inhibit ATP-mediated lysosomal acidification, inhibit ATPstimulated cystine transport in human lymphoblasts, but have had no effect on transport in other studies (Gahl et al, 1983(Gahl et al, , 1984aJonas et al, 1982bJonas et al, , 1983. In all of these studies, cystine transport was examined in only partially purified lysosomal preparations (Steinherz et al, 1982a;Gahl et al, 1982aGahl et al, ,b, 1983Gahl et al, , 1984aJonas et al, 1982aJonas et al, ,b, 1983Smith et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Cystine transport in purified rat liver lysosomes

Jonas¹

1986

Biochemical Journal

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Amino acid efflux from highly purified rat liver lysosomes exposed to the methyl ester derivatives of leucine, methionine, tyrosine and cystine was examined. The lysosomal efflux of leucine, methionine and tyrosine was unaffected by the presence of MgATP, whereas cystine efflux was stimulated by MgATP. Exposure of lysosomes to 2 mM-MgATP resulted in lysosomal acidification and a 0.5 pH unit increase in the lysosomal pH gradient through the action of a proton-pumping ATPase. Cystine efflux was also stimulated when the lysosomal proton gradient was increased through changes in buffer pH. Decreasing the lysosomal proton gradient with ionophores resulted in diminished cystine efflux. Bivalent cations had no effect on the lysosomal efflux of leucine, methionine and tyrosine. However, cystine efflux was stimulated by the presence of bivalent cations even when the lysosomal proton gradient was minimized. Cation-stimulated cystine efflux was inhibited by the presence of the calcium ionophore A23187, which altered the lysosomal membrane potential. Cystine efflux from lysosomes appears to be uniquely dependent on pH gradients and cation concentrations.

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The Role of Atp in Lysosomal Cystine Efflux

Cited by 7 publications

References 0 publications

Demonstration of adenosine deaminase activity in human fibroblast lysosomes

Demonstration of adenosine deaminase activity in human fibroblast lysosomes

Lysosomal pH: a link between cell volume and metabolism

Cystine transport in purified rat liver lysosomes

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