Bazzi M D, Nelsestuen G L
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1990 Aug 21;29(33):7624-30. doi: 10.1021/bi00485a012.
The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.
在有磷脂和无磷脂存在的情况下，通过平衡透析研究了钙和磷脂依赖性蛋白激酶C（PKC）的钙结合特性。钙与PKC的结合表现出惊人且出乎意料的行为；在细胞内钙浓度下，游离的PKC几乎不结合钙，而在200微摩尔/升钙浓度下结合有限量的钙（约每摩尔PKC结合1摩尔钙）。然而，在含有酸性磷脂的膜存在时，每个PKC蛋白至少结合8个钙离子。透析缓冲液中1微摩尔佛波醇二丁酸酯（PDBu）的存在对这些钙结合特性影响很小。在平衡条件下通过凝胶过滤分析PKC与钙的结合得到了类似的结果；只有与膜结合的PKC结合大量的钙。因此，PKC是可能以磷脂依赖性方式结合钙的一大类蛋白质中的一员。诱导PKC与膜结合所需的钙浓度与钙结合所需的浓度相似（中点时约为40微摩尔/升钙）。然而，PKC与膜结合所需的钙浓度受到膜中磷脂酰丝氨酸组成的强烈影响。磷脂酰丝氨酸百分比更高的膜需要更低的钙浓度。这些特性表明钙结合位点可能在PKC与膜的界面处产生。钙可能作为PKC与磷脂之间的桥梁。这些研究还表明，除了钙水平和膜中二酰基甘油含量外，钙依赖性PKC与膜的结合以及PKC的功能可能受多种因素调节。
