67 kDa calcimedin, a new Ca2+-binding protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Moore PB
PROVIDER: S-EPMC1147096 | biostudies-other | 1986 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The 67 k calcimedin is a Ca2+-binding protein present in both muscle cells and peritoneal macrophages. Many tissues, including lymphoid tissues, liver and lymphocytes, have been shown to contain Ca2+-binding proteins of similar molecular size, such as the p67(67 kDa) calelectrin or the 68 kDa lymphocyte protein. We have tested affinity-purified antibodies raised to the smooth-muscle 67 k calcimedin in these several tissues and here report that the 67 k calcimedin is not detectable in liver, thymus, spleen or thymic lymphocytes. These findings support recent biochemical evidence, discussed here, suggesting that the 67 k calcimedin is a protein different from calelectrin and the 68 kDa lymphocyte protein. The more limited tissue distribution of the 67 k calcimedin, which includes muscle and macrophages, suggests that the 67 k calcimedin may function in Ca2+-mediated events special to these cell types. The affinity-purified antibodies to the 67 k calcimedin will be useful in obtaining information concerning the special roles of this Ca2+-binding protein in these cells.
| S-EPMC1148979 | biostudies-other
Project description:The 67 kDa calcimedin, isolated by using a phenyl-Sepharose affinity column followed by DEAE-cellulose and gel-filtration chromatographies, was homogeneous by the criterion of SDS/polyacrylamide-gel electrophoresis. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other Ca2+-binding proteins such as calmodulin and S-100 proteins. The 67 kDa protein underwent a conformational change upon binding Ca2+, as revealed by u.v. difference spectroscopy and near-u.v. c.d. measurements. Tryptophan and tyrosine residues were perturbed upon Ca2+ binding, moving to a more non-polar environment in the presence of Ca2+. Upon excitation of the protein at 280 nm, the fluorescence emission maximum was centered around 325 nm, suggesting that the tryptophan residues are located in a fairly hydrophobic region. Ca2+ addition did not induce a significant change in the intrinsic protein fluorescence intensity at 325 nm. Addition of Ca2+ to the 67 kDa protein labelled with 2-p-toluidinylnaphthalene-6-sulphone (TNS) resulted in a 25% increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 442 to 432 nm. Hence, the probe in the presence of Ca2+ moves to a more non-polar microenvironment, like calmodulin and other Ca2+-binding proteins. Fluorescence titration with Ca2+ using TNS-labelled protein revealed one class of binding site, with a Kd value of 2 x 10(-5) M.
| S-EPMC1138588 | biostudies-other
Project description:The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrP(C)) and to be required for pathological PrP (PrP(Sc)) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrP(C) interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrP(C) in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrP(C). Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrP(C) interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrP(C) on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrP(C) and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases.
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Project description:A Ca2+-binding protein of molecular mass 48 kDa and named 'CAB-48' has been purified from bovine brain 100,000 g supernatant. About 30 mg of CAB-48 was purified from 1 kg of bovine brain. The protein has been characterized with respect to its physical, chemical and Ca2+-binding properties. It has an apparent molecular mass of 48 kDa by SDS/polyacrylamide-gel-electrophoresis and 75.2 kDa from sedimentation-velocity and Stokes-radius data. The acidic nature of the molecule is suggested by its pI of 4.7. In the presence of 3.0 mM-MgCl2 and 150 mM-KCl, CAB-48 binds 1.0 mol of Ca2+/mol of protein with an apparent Kd of 15 microM. A tyrosine protein kinase partially purified from rat spleen catalysed the incorporation of 0.73 mol of phosphate/mol of CAB-48, and phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues.
| S-EPMC1148240 | biostudies-other
Project description:The 67 kDa calcimedin is a Ca2+-binding protein isolated from several muscle tissues. A recent report [Morse & Moore (1988) Biochem. J. 251, 171-174] indicated that the 67 kDa calcimedin is distinct from 67 kDa calelectrin, which is purified from various non-muscle cells. In the present study we have purified the 67 kDa protein from bovine aorta (i.e. 67 kDa calcimedin) and liver (i.e. 67 kDa calelectrin) and compared them by immunological and biochemical criteria. The aorta calcimedin is identical with the liver calelectrin by the following criteria. (1) The calcimedin co-electrophoresed with the calelectrin on SDS/5-15%-(w/v)-linear-gradient polyacrylamide gels. (2) The two proteins selectively cross-reacted with a chicken gizzard calcimedin antibody. (3) An antibody raised against the bovine aorta calcimedin also recognized the bovine liver calelectrin. (4) One-dimensional peptide maps of the two proteins revealed no significant difference. (5) The calcimedin appeared to have an amino acid composition essentially the same as that of the liver calelectrin. (6) The amino acid sequences of the calcimedin fragments were identical with those of the calelectrin fragments.
| S-EPMC1133373 | biostudies-other
Project description:The 67 kDa laminin receptor (67LR) is a non-integrin receptor for laminin (LM) that derives from a 37 kDa precursor (37LRP). 67LR expression is increased in neoplastic cells and correlates with an enhanced invasive and metastatic potential. We used structure-based virtual screening (SB-VS) to search for 67LR inhibitory small molecules, by focusing on a 37LRP sequence, the peptide G, able to specifically bind LM. Forty-six compounds were identified and tested on HEK-293 cells transfected with 37LRP/67LR (LR-293 cells). One compound, NSC47924, selectively inhibited LR-293 cell adhesion to LM with IC50 and Ki values of 19.35 and 2.45 μmol/L. NSC47924 engaged residues W176 and L173 of peptide G, critical for specific LM binding. Indeed, NSC47924 inhibited in vitro binding of recombinant 37LRP to both LM and its YIGSR fragment. NSC47924 also impaired LR-293 cell migration to LM and cell invasion. A subsequent hierarchical similarity search with NSC47924 led to the identification of additional four compounds inhibiting LR-293 cell binding to LM: NSC47923, NSC48478, NSC48861, and NSC48869, with IC50 values of 1.99, 1.76, 3.4, and 4.0 μmol/L, respectively, and able to block in vitro cancer cell invasion. These compounds are promising scaffolds for future drug design and discovery efforts in cancer progression.
| S-EPMC4627239 | biostudies-literature
Project description:The 37/67-kDa laminin receptor (LAMR/RPSA) was originally identified as a 67-kDa binding protein for laminin, an extracellular matrix glycoprotein that provides cellular adhesion to the basement membrane. LAMR has evolutionary origins, however, as a 37-kDa RPS2 family ribosomal component. Expressed in all domains of life, RPS2 proteins have been shown to have remarkably diverse physiological roles that vary across species. Contributing to laminin binding, ribosome biogenesis, cytoskeletal organization, and nuclear functions, this protein governs critical cellular processes including growth, survival, migration, protein synthesis, development, and differentiation. Unsurprisingly given its purview, LAMR has been associated with metastatic cancer, neurodegenerative disease and developmental abnormalities. Functioning in a receptor capacity, this protein also confers susceptibility to bacterial and viral infection. LAMR is clearly a molecule of consequence in human disease, directly mediating pathological events that make it a prime target for therapeutic interventions. Despite decades of research, there are still a large number of open questions regarding the cellular biology of LAMR, the nature of its ability to bind laminin, the function of its intrinsically disordered C-terminal region and its conversion from 37 to 67 kDa. This review attempts to convey an in-depth description of the complexity surrounding this multifaceted protein across functional, structural and pathological aspects.
| S-EPMC5249262 | biostudies-literature
Project description:It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.
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Project description:We surveyed rabbit brain cytosol for a new Ca2+/calmodulin (CaM)-dependent kinase. The renaturation blotting assay (RBA) exploits the ability of blotted SDS-denatured proteins to regain enzymic activity after guanidine treatment. Using RBA, we found that the eluate of rabbit brain cytosol from a CaM affinity column contains at least four electrophoretically distinct protein kinase bands which were autophosphorylated in a Ca2+/CaM-dependent manner. The 49 kDa band and the 60 kDa band were alpha and beta subunit of CaM kinase II, and the 42 kDa band was presumed to be CaM kinase I, but the 80 kDa band could not be attributed to any reported Ca2+/CaM-dependent protein kinases. The 80 kDa protein kinase was isolated by three-step chromatography. We examined the phosphorylation of exogenous substrates by 80 kDa protein kinase, and histone IIIs and myosin light chain were phosphorylated in a Ca2+/CaM-dependent manner. W-7, a specific inhibitor for calmodulin, inhibited this kinase activity, but KN-62, a specific inhibitor for CaM kinase II, had no effect on this protein kinase activity. Autoradiography using boiled rabbit brain homogenate as substrate showed three intrinsic substrates (80 kDa, 60 kDa and 42 kDa), which were phosphorylated in a Ca2+/CaM-dependent manner. These findings suggest that a new Ca2+/CaM-dependent protein kinase could be identified by the RBA.
| S-EPMC1130688 | biostudies-other
Project description:The 67LR (67 kDa laminin receptor) enables cells to interact with components of the extracellular matrix. The molecule is derived from the 37LRP (37 kDa laminin receptor precursor); however, the precise molecular mechanism of this conversion is unknown. Recombinant 37LRP, expressed in and purified from Escherichia coli, bound to human laminin in a SPR (surface plasmon resonance) experiment. 67LR isolated from human breast-cancer-derived cells in culture was also shown to bind to laminin by SPR. However, the kinetics of association are qualitatively different. 37LRP, but not 67LR, binds to heparan sulfate. The binding of 37LRP to heparan sulfate did not affect the interaction of 37LRP with laminin. In contrast, heparan sulfate reduces the extent of binding of laminin to 67LR. Taken together, these results show that 37LRP has some of the biological activities of 67LR, even prior to the conversion event. However, the conversion affects the sites of interaction with both laminin and heparan sulfate.
| S-EPMC2782360 | biostudies-literature