Demonstration of adenosine deaminase activity in human fibroblast lysosomes

Abstract

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.

Identification of a Novel Membrane Transporter Associated with Intracellular Membranes by Phenotypic Complementation in the Yeast Saccharomyces cerevisiae

“…A requirement for intracellular transport of nucleosides is predicted by the compartmentalization of enzymes of nucleoside metabolism within various organelles (41)(42)(43). Mediated transport of nucleosides has been demonstrated in lysosomes isolated from human fibroblasts (44), and uptake of nucleosides has been observed in isolated mitochondria (45).…”

Inhibition of Transient Receptor Potential Channel Mucolipin-1 (TRPML1) by Lysosomal Adenosine Involved in Severe Combined Immunodeficiency Diseases

“…Lysosomes-Because ADA activity has been detected in lysosomes (23,24), loss of ADA presumably leads to adenosine accumulation in lysosomes. To explore the role of adenosine in lysosomes, we first measured the level of adenosine in lysosomes isolated from human skin fibroblasts using high performance liquid chromatography (HPLC) (25).…”

“…It is very likely that ENT3-mediated adenosine release can replenish cytosolic adenosine, which is important for many intracellular processes under normal physiological condition. We can also speculate that lysosomal adenosine may be released into the cytosol to participate in ATP synthesis, especially during nutritional starvation (23). Lysosomes are dynamic organelles that constitutively fuse with the plasma membrane to release luminal substances, so-called lysosomal exocytosis.…”

“…Because 5Ј-nucleotidase, the enzyme that dephosphorylates AMP to generate adenosine, has been found in lysosomes (51,52), it is likely that ATP breakdown in the lysosomal interior acts as an alternative pathway that generates adenosine. In addition, lysosomal adenosine homeostasis is maintained not only by the biogenesis pathway but also by the degradation pathway, which may be dominated by lysosomal ADA (23,24), and the release pathway mediated by ENT3 (7). In agreement with this, the loss of either ADA (this study) or ENT3 (7) causes lysosomal adenosine accumulation.…”

“…These phenocopy mice with deficiency in transient receptor potential channel mucolipin-1 (TRPML1), a lysosome Ca 2ϩ -permeable channel regulating lysosomal membrane trafficking, lysosome function, and apoptotic cell clearance (16 -22). Interestingly, ADA activity has been detected in lysosomes isolated from human fibroblasts (23,24).…”