Purification and characterization of ATP-dependent calcium pumps from synaptosomes.
Purification and characterization of ATP-dependent calcium pumps from synaptosomes.
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突触体中 ATP 依赖性钙泵的纯化和表征。
DOI：
10.1101/sqb.1983.048.01.031
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
H. Rahamimoff
中科院分区：
文献类型：
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作者：
S. M. Goldin;S. Chan;D. Papazian;E. Hess;H. Rahamimoff
引发神经递质释放的主要事件是神经末梢细胞质中Ca++浓度的增加（综述见Kelly et al. 1979）。神经递质释放通常通过细胞质Ca+水平的降低而终止。因此，Ca++在神经递质释放的起始和终止中的作用类似于其在肌肉收缩的起始和终止中的作用（综述见Carafoli和Crompton 1978）。负责肌肉收缩的Ca++通量受多种机制调节;例如，负责从心肌细胞的肌浆中清除Ca++的肌浆网Ca+泵可受cAMP（Tada et al. 1979）调节，以响应儿茶酚胺，并同时受钙调蛋白（LePeuch et al. 1979）调节。这些形式的调节涉及通过与Ca++泵相关的调节蛋白的至少两种不同的蛋白激酶的磷酸化。在过去的十年中，我们对肌肉功能调节的分子基础的理解有了显著的进步，部分原因是对所涉及的Ca++泵的鉴定和纯化（MacLennan和Holland 1975），以及随后在体外通过钙调蛋白和cAMP对其调节的证明。有证据表明，突触体（一种富含来自脑匀浆的神经末梢的制剂）含有ATP依赖性Ca+泵（Morgan 1976）。这种泵可能通过挤出穿过质膜和/或通过将Ca++隔离在末端的囊泡结构内而从神经末端的细胞质中除去Ca++。与肌肉收缩的调节类似，作用于突触前受体的药物和内源性物质（如阿片类药物和脑啡肽）对神经递质释放的调节可能涉及负责神经末梢中Ca++转运的蛋白质活性的生化修饰（Mudge et al. 1979; Ross and Cardenas 1979）。Ca++泵在调节神经递质释放中的潜在重要性是其鉴定、纯化和表征的强烈动机。本文总结了我们在这方面取得的进展。我们一直在应用生物化学和免疫学技术研究哺乳动物脑突触体中ATP依赖的Ca++转运，考虑到以下问题。在神经末梢中有多少个分子上不同的Ca++泵？这些泵是否与其他哺乳动物细胞的Ca++泵相同，例如肌浆网（MacLennan and Holland 1975）和红细胞（Niggli et al. 1979）的Ca++泵，或者它们是神经细胞特有的？它们的功能主要是从神经末梢中挤出Ca++还是将Ca++隔离在其中？除了通过Ca++和ATP的浓度进行初级调节外，它们是否还受到次级机制的调节？如果它们受到调节，那么这些机制的生理作用是什么？
The primary event that initiates the release of neurotransmitter is an increase in the Ca++ concentration in the nerve terminal cytoplasm (for review, see Kelly et al. 1979). Neurotransmitter release is normally terminated by reduction in cytoplasmic Ca § + levels. Thus, the role of Ca++ in the initiation and termination of neurotransmitter release is analogous to its role in initiation and termination of muscle contraction (for review, see Carafoli and Crompton 1978). Ca++ fluxes responsible for muscle contraction are regulated by various mechanisms; eg, the sarcoplasmic reticulum Ca+ § pump, responsible for Ca++ removal from the myoplasm of cardiac muscle cells, can be regulated by cAMP (Tada et al. 1979) in response to catecholamines and, in parallel, by calmodulin (LePeuch et al. 1979). These forms of regulation involve phosphorylation by at least two distinct protein kinases of a regulatory protein associated with the Ca++ pump. Our understanding of the molecular basis of the regulation of muscle function has advanced significantly in the last decade, due in part to the identification and purification of the Ca++ pump involved (MacLennan and Holland 1975) and to the subsequent demonstrations in vitro of its regulation by calmodulin and cAMP. Evidence exists that synaptosomes, a preparation enriched in nerve terminals derived from brain homogenates (Morgan 1976), contain ATP-dependent Ca § + pumps. Such pumps presumably remove Ca++ from the cytoplasm of the nerve terminal by extrusion across the plasma membrane and/or by sequestration of Ca++ within vesicular structures in the terminals. By analogy with the regulation of muscle contraction, regulation of neurotransmitter release by drugs and endogenous substances (eg, the opiates and enkephalins) that act at presynaptic receptors may involve biochemical modification of the activity of proteins responsible for Ca++ transport in nerve terminals (Mudge et al. 1979; Ross and Cardenas 1979). The potential importance of the Ca++ pumps in the regulation of neurotransmitter release is a strong motive for their identification, purification, and characterization. This paper summarizes the progress we have made in this area. We have been applying biochemical and immunological techniques to the study of ATP-dependent Ca++ transport in synaptosomes of mammalian brain, with the following questions in mind. How many molecularly distinct Ca++ pumps are there in nerve terminals? Are these pumps identical to the Ca++ pumps from other mammalian cells, such as the Ca++ pumps of sarcoplasmic reticulum (MacLennan and Holland 1975) and erythrocyte (Niggli et al. 1979), or are they specific to nerve cells? Is their function primarily to extrude Ca++ from the nerve terminal or to sequester Ca++ within it? Are they regulated by secondary mechanisms in addition to their primary regulation by the concentrations of Ca++ and ATP? If they are regulated, what is the physiological role of such mechanisms?