Covalent labeling of the cytoplasmic or luminal domains of the sarcoplasmic reticulum Ca(2+)-ATPase with fluorescent azido dyes.
Sarcoplasmic reticulum (SR) vesicles were incubated with azido derivatives of Cascade blue (ACB), Lucifer yellow (ALY), 2,7-naphthalene-disulfonic acid (ANDS), and fluorescein (AF) for 0.1-24 h at 2 degrees C. All four dyes gave intense reaction with the cytoplasmic domain of the Ca(2+)-ATPase on photoactivation after brief incubation. The penetration of the dyes into the luminal space of the SR was determined after centrifugation through Sephadex microcolumns to remove the external dye, followed by photolabeling and gel electrophoresis of the photolabeled proteins. The reaction of ACB and ANDS with the Ca(2+)-ATPase and with calsequestrin increased progressively during incubation up to 24 h indicating their slow accumulation in the luminal space, while ALY and AF did not show significant penetration into the vesicles. The distribution of the covalently attached ACB in the Ca(2+)-ATPase was tested by tryptic proteolysis after labeling exclusively from the outside (OS), from the inside (IS) or from both sides (BS). In all cases intense ACB fluorescence was seen in the A fragment with inhibition of ATPase activity. In the OS preparations the A1, while in IS the A2 fragment was more intensely labeled. There was no significant incorporation of ACB into the region of B fragment identified by FITC fluorescence. The crystallization of the Ca(2+)-ATPase by EGTA + decavanadate was completely inhibited in the BS samples after labeling either in the Ca2E1 or E2V conformation. There was no inhibition of crystallization in the OS preparations. In the IS preparations labeled in the Ca2E1 state the crystallization was impaired, while in the E2V state there was only slight disorganization of the crystals. The total amount of ACB photoincorporated into SR proteins after incubation for 24 h was 1.75 nmol/mg protein; 2/3 of this labeling occurred from the outside and 1/3 from the inside. Similar level of labeling was obtained in media that stabilize the E1 or the E2 conformation of the Ca(2+)-ATPase.
Molnar
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E
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210.
Varga
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S
Jona
I
I
Martonosi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007425:Intracellular Membranes / Q000201:enzymology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000276:immunology; D009943:Organophosphorus Compounds / Q000276:immunology; D010446:Peptide Fragments / Q000302:isolation & purification; D010777:Photochemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure; D013861:Thiocyanates; D014357:Trypsin; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology
D000906:Antibodies; D005452:Fluoresceins; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D013861:Thiocyanates; C066850:Cascade Blue; D014638:Vanadates; C032826:azidofluorescein; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate