The photoreactive ADP analogue 8-N3-ADP binds in the dark to the catalytic site of the sarcoplasmic reticulum Ca-ATPase. An apparent Kd value of 30 microM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[3H]ADP in the presence of calcium results in irreversible inhibition of ATPase activity with corresponding stoichiometries of covalently and specifically photolabeled Ca-ATPase. The site of photolabeling of the Ca-ATPase in the presence of calcium has been explored. Controlled trypsin digestion of the labeled protein shows that 8-azido-ADP is incorporated in the B subfragment. Extensive trypsin digestion of the labeled protein releases a small peptide as revealed by gel filtration chromatography (Sephadex G-50). Further HPLC purification on a reverse-phase column (C8) eluted with a water/acetonitrile gradient buffered at pH 6 or at pH 2 gives a single labeled peptide. Edman degradation of that isolated peptide, as well as the amino acid composition, shows that it contains five amino acid residues (Val-530-Arg-534) with the radioactivity localized on Thr-532 and Thr-533.
Nucleotidase activity and Ca-uptake were characterized in
endoplasmic reticulum (ER) enriched rat submandibular gland
(SMG) microsomal preparations. (i) Ca-uptake had characteristics
of an ER Ca-ATPase. (ii) Nucleotidase activity was equally
stimulated by calcium, magnesium and manganese, but with
different Km values. (iii) Specific inhibitors of P-type Ca-ATPases
were ineffective on nucleotidase activity, demonstrating that this
activity was not related to calcium uptake and did not correspond
to classical Ca2+ pumps. (iv) ATP and UTP were more efficient
substrates, whereas ADP and UDP were hydrolyzed at
significantly slower rate. (v) Nucleotidase activity was sensitive to
mild detergent solubilization and insensitive to ionophore
addition. (vi) Nucleotidase activity was strongly inhibited by
suramin, a nucleoside triphosphate diphosphohydrolase
(NTPDase) inhibitor. (vii) Nucleotidase activity exponentially
diminished as function of time. All these observations are
consistent with a NTPDase identity. The presence of a NTPDase
was demonstrated by immunohistochemistry in rat SMG.
Immunoreactivity was stronger in ductal cells than in mucous and
serous acini. Although this enzyme was observed in the plasma
membrane, colocalization with the ER marker calnexin revealed a
specific subcellular localization in this organelle of all three types
of cell. The putative function of this NTPDase activity in salivary
glands is discussed.
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