Schultz J E, Uhl D G, Klumpp S
Pharmazeutisches Institut der Universität, Tübingen, Federal Republic of Germany.
Biochem J. 1987 Aug 15;246(1):187-92. doi: 10.1042/bj2460187.
The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.
对与四膜虫可兴奋纤毛膜相关的腺苷酸环化酶的离子调节动力学进行了研究。甘油（30%，v/v）可使该酶稳定，并通过增加Vmax（3倍）和降低MgATP的表观Km（6倍）来激活它。对Mg2+作用的动力学分析表明，它通过一个与底物位点分开的单一金属结合位点起刺激作用，解离常数Ks为0.27 mM。对Ca2+作用的分析表明：（i）对底物MgATP存在非竞争性抑制；（ii）抑制程度取决于游离Mg2+浓度。在分别存在0.55 - 2 mM - Mg2+的情况下，Ki值范围为4至130 μM - Ca2+。这表明Mg2+和Ca2+在金属结合位点存在竞争。Ca2+的作用具有特异性；Sr2+和Ba2+几乎无作用，100 μM - Ba2+不干扰Ca2+的抑制作用。加入EGTA后，Ca2+的作用很容易逆转。K+在浓度约为20 mM时激活腺苷酸环化酶。K+的刺激效力取决于游离Mg2+浓度。在1 mM游离Mg2+时，20 mM - K+使腺苷酸环化酶活性增加一倍。抑制性的Ca2+输入和刺激性的K+输入相互独立。
