Calcium binding proteins isolation

Cormier, M. J., and Charbonneau, H. (1977). Isolation, properties and function of a calcium-triggered luciferin binding protein. In Wasserman, R. H., et al. (eds.), Calcium Binding Proteins and Calcium Function, pp. 481-489. Elsevier North-Holland. 

Sorcin (soluble resistance-related calcium binding protein) was isolated from multidrug-resistant cells and is expressed in a few mammalian tissues such as skeletal muscle, heart, and brain. In the heart, sorcin interacts with the ryanodine receptor and L-type Ca2+-channels regulating excitation in contraction coupling. 

It has been shown that the activity of NO synthases is regulated by cofactors calcium binding protein calmodulin and tetrahydrobiopterin (H4B). Abu-Soud et al. [149] have studied the effect of H4B on the activity of neuronal nNOS I, using the isolated heme-containing oxygenase domain nNOSoxy. It was found that nNOSoxy rapidly formed an oxygenated complex in the reaction with dioxygen, which dissociated to produce superoxide (Reaction (6)) ... 

A protein of similar molecular weight to that of rat oncomodulin, rat and rabbit parvalbumins, S100, and the vitamin D-dependent calcium-binding proteins has been isolated from chicken gizzard smooth muscle. In this case, however, the fluorescence emission from the four tyrosine residues is quenched by Ca2+ binding.(160) The decrease in fluorescence intensity was used to suggest that there are two different classes of Ca2+binding sites. 

A Ca +-binding protein isolated from jellyfish Aequorea sp.) that is frequently used as a chemiluminescent calcium indicator.Aequorin contains a hydrophobic prosthetic group, coelenterazine. Investigators have been able to express the transfected gene recombinantly in a number of cells, and upon addition of the cofactor, one can measure intracellular calcium concentration. See Fura-2... 

A variety of other calcium transport systems are associated with Ca21-activated ATPases. The extraembryonic structure, the chorioallantoic membrane, of the chick embryo is responsible for the translocation of over 120 mg of eggshell calcium into (he embryo during development. The enzyme responsible for this is a (Ca2+, Mg2+)-ATPase with Km values for Ca2+ of 30 p,mol dm-3 and 0.3 mmol dm-3, and a molecular weight of 170 000. The enzyme can be crossiinked and co-isolated with a calcium-binding protein.158 Transport of Ca2+ is also associated with (Ca2+, Mg2+)-ATPases in neutrophil plasma membranes,159 transverse tubule membranes from rabbit skeletal muscle,160 rabbit myocardial membrane,161 endoplasmic reticulum,162 sar-colemma,163 brain microsomes,164 the Golgi apparatus165 and rat liver plasma membranes.166... 

A wide range of calcium-binding proteins have been isolated from intracellular and extracellular sources. 11S 119 209,210 They are involved in the storage of calcium, the buffering of calcium concentrations and the triggering of a wide range of processes involving membrane transport, secretion... 

The last calcium binding protein to be discussed in the miscellaneous category is calsequestrin. This protein was isolated from the lumen of the sarcoplasmic reticulum and because of the high binding capacity, has been suggested to be a major site of calcium binding in the interior of the sarcoplasmic reticulum. 

Retinal calcium-binding proteins obtained from the phenyl-Sepharose column can be separated by RP-HPLC (20). Calmodulin elutes first, followed by Capl. S-1006 elutes as two distinct peaks, while recoverin was separated into a series of at least three overlapping peaks (20). Mass spectrometry revealed that these peaks correspond to the different fatty acid modified forms of recoverin. The first peak contains the C14 2 and C12 0 acylated forms. The second recoverin peak consists of the C14 l species and the third peak C14 0. The same procedure may be used to isolate similarly modified proteins, including the photoreceptor G protein transducin. 

DEAE-Sepharose in low calcium. In addition to these relatively soluble calcium-binding proteins, other less soluble EF hand proteins (eg. GCAP) can be purified by relatively simple procedures. Such isolation procedures will be of increasing importance for studying signal transduction pathways that involve calcium. 

A wide range of calcium-binding proteins have been isolated from intracellular and extracellular... 

The localization of the IP, receptor has been compared to the localization of other luminal or membrane components of the endoplasmatic reticulum related to Ca "" homeostasis. The membrane pump Ca -ATPase, immunolabelled with antibodies against cardiac Ca -ATPase, was found to be located in regular cisternae of the endoplasmatic reticulum, the lateral tips of cisternae of the Golgi complex and in calciosomes of Purkinje cells (Kaprielian, 1989 Michelangeli et al., 1991 Villa et al., 1991 Takei et al., 1992) (Fig. 27). Distal axons of Purkinje cells, however, lacked Ca "-ATPase im-munoreactivity (Takei et al., 1992). Appreciable levels of calsequestrin, the main intraluminal calcium-binding protein of muscle, were present in Purkinje cells of the chicken. Calsequestrin-immunoreactivity was present over the lumen (Villa et al., 1991) and membranes (Takei et al., 1992) of stacked and isolated cisternae of the endoplasmatic reticulum and in a subpopulation of calciosomes (Volpe et al., 1988 Volpe and Villa,... 

The calcium-transport system is another potential source of binding proteins. Transport into mitochondria, across plasma membranes and through the intestinal mucosa and the renal tubule may be the function of distinct transport systems, each of which may be the source of binding proteins. The possibility of isolating bacterial mutants deficient both in calcium uptake and in calcium-binding proteins offers an interesting approach to the role of binding proteins in calcium transport. 

An intestinal Vitamin D-inducible calcium binding protein was found in monkeys (128)] this protein, unlike those from the dog and cow, has a lower electrophoretic mobility than the chick protein, but no chemical data other than this observation have been reported. Reports on the presence of two calcium-binding proteins from human intestinal mucosa (129, 130) are very preliminary and aside from the indication that one of the proteins has a molecular weight of about 20,000 they provide little solid information. The same or a similar protein was also isolated from human kidney (131), and it appears probable that the calcium-binding protein recently found in lactating bovine and rat mammary gland (132) s identical to the intestinal protein. 

A calcium-binding protein has recently been isolated from the optic lobes of squid and cuttlefish by Alema and his co-workers (161b). 

Strontium ion was found to support the asynchronous mode of transmitter release in isolated layer V pyramidal cells of the prefrontal cortex (Aghajanian and Marek 1999). This is apparently mediated through calcium-binding protein synaptotagmin III, as strontium does not support the function of calciumbinding synaptotagmins I and II (Li et al. 1995). 

In our laboratory, we have developed a new protocol for isolating four calcium binding proteins from smooth muscle. These include 67-kDa calcium binding protein (67 calcimedin), calmodulin, 12-kDa calcium... 

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