CHARACTERIZATION OF A PUTATIVE CA2+-TRANSPORTING CA2+-ATPASE IN THE PELLICLES OF PARAMECIUM-TETRAURELIA

被引:38
作者
WRIGHT, MV [1 ]
VANHOUTEN, JL [1 ]
机构
[1] UNIV VERMONT, DEPT ZOOL, BURLINGTON, VT 05405 USA
关键词
(Paramecium); Calcium pump; Phosphoenzyme intermediate; Plasma membrane;
D O I
10.1016/0005-2736(90)90160-P
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In Paramecium, no Ca2+-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2+-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2+-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 μM). Vanadate and calmidazolium inhibit Ca2+-stimulated activity with an EC50 of about 2 μM and 0.5 μM, respectively. Likewise, 10 μM trifluoperazine inhibits 80% of Ca2+-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2+-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 μg/ml) or ouabain (0.2 mM). Incubation of pellicles with [γ-32P]ATP specifically labels a 133 kDa protein in a Ca2+-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 μM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2+-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2+-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed < 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 μM) compared to the total Ca2+-ATPase activity. Therefore, the majority of the Ca2+-ATPase activity is likely to be plasma membrane associated. © 1990.
引用
收藏
页码:241 / 251
页数:11
相关论文
共 60 条
[1]   BIOCHEMICAL-STUDIES OF THE EXCITABLE MEMBRANE OF PARAMECIUM-TETRAURELIA .3. PROTEINS OF CILIA AND CILIARY MEMBRANES [J].
ADOUTTE, A ;
RAMANATHAN, R ;
LEWIS, RM ;
DUTE, RR ;
LING, KY ;
KUNG, C ;
NELSON, DL .
JOURNAL OF CELL BIOLOGY, 1980, 84 (03) :717-738
[3]  
ANDRIVON C, 1983, BIOL CELL, V47, P351
[4]  
Aronson N N Jr, 1974, Methods Enzymol, V31, P90
[5]   VANADATE INHIBITION OF THE CA2+-ATPASE FROM HUMAN RED-CELL MEMBRANES [J].
BARRABIN, H ;
GARRAHAN, PJ ;
REGA, AF .
BIOCHIMICA ET BIOPHYSICA ACTA, 1980, 600 (03) :796-804
[6]  
BILINSKI M, 1981, EUR J CELL BIOL, V24, P108
[7]   ELECTROPHYSIOLOGICAL STUDY OF REGULATION OF CILIARY BEATING FREQUENCY IN PARAMECIUM [J].
BREHM, P ;
ECKERT, R .
JOURNAL OF PHYSIOLOGY-LONDON, 1978, 283 (OCT) :557-568
[8]   BIOCHEMICAL STUDIES OF EXCITABLE MEMBRANE OF PARAMECIUM-AURELIA .1. CA-45(2+) FLUXES ACROSS RESTING AND EXCITED MEMBRANE [J].
BROWNING, JL ;
NELSON, DL .
BIOCHIMICA ET BIOPHYSICA ACTA, 1976, 448 (02) :338-351
[9]  
BRUGEROLLE G, 1980, BIOL CELLULAIRE, V37, P251
[10]  
CARONI P, 1981, J BIOL CHEM, V256, P3263