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28 kDa adenosine-binding proteins of brain and other tissues.

ABSTRACT: Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.

SUBMITTER: Ravid K

PROVIDER: S-EPMC1138416 | biostudies-other | 1989 Mar

REPOSITORIES: biostudies-other

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Project description:Intracellular free calcium ions (Ca(2+)) are an important element in retinal ganglion cell response. Two major EF-hand (E-helix-loop-F-helix-hand) calcium binding proteins in the retina, calretinin and calbindin-28 kDa, are important buffers of intracellular free Ca(2+) in neurons, and may also serve as Ca(2+)-dependent regulators of enzymes and ion channels.This study used immunohistochemistry to investigate the subcellular expression patterns of calretinin and calbindin-28 kDa, in the soma, dendrites, and the axonal compartment of rat retinal ganglion cells.Antibodies for calretinin and calbindin-28 kDa labeled different cell populations in the retinal ganglion cell layer. In this layer, calretinin labeled a larger number of cells compared to calbindin-28 kDa, many, but not all, of which were displaced amacrine cells. The calbindin-28 kDa immunopositive neurons were distinct in that their somata were peripherally encircled by microtubule associated protein 1 (MAP-1) or neurofilament-200 kDa subunit (NF-200 kDa) immunofluorescence. Although somata of retinal ganglion cells contained these calcium binding proteins, neither protein was found in the dendrites or initial segments of the axons. However, both were expressed in the ganglion cell axons in nerve fiber layer. Calretinin and calbindin-28 kDa staining overlapped in some fibers and not in others. Calretinin immunofluorescence was concentrated in discrete axonal regions, which showed limited staining for calbindin-28 kDa or for NF200 kDa, suggesting its close proximity to the plasma membrane.There is a clear compartmentalization of calbindin-28 kDa and calretinin distribution in retinal ganglion cells. This suggests that the two calcium binding proteins perform distinct functions in localized calcium signaling. It also indicates that rather than freely diffusing through the cytoplasm to attain a homogeneous distribution, calbindin-28 kDa and calretinin must be bound to cellular structures through interactions that are likely important for their functions.

Project description:1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.

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28 kDa adenosine-binding proteins of brain and other tissues.

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