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Biomedical subjects

A L de Leeuw

Publications and source records attributed to A L de Leeuw.

12 recordsLinked to original sources
PubMed1986

Calcium-binding lens membrane proteins.

Calcium-binding membrane-bound proteins are present in the vertebrate eye lens. Among these proteins are a distinct group of immunologically related extrinsic EDTA-extractable proteins (EEP) and calmodulin. The EEP proteins contain calcium-binding sites with a total capacity of 25 mol Ca2+ per mol protein. This high calcium-binding capacity of EEP points to a function of these proteins as intracellular calcium store in the lens. However, EEP undergoes a conformational change upon calcium binding, indicating that these proteins may be involved in the regulation of calcium-dependent cellular processes in the lens. One of these processes is the action of communicating lens fiber junctions, which contain EEP as a main protein component. In addition to EEP, another calcium-binding protein in lens, calmodulin, probably functions as mediator of calcium in the regulation of the structure and function of lens junctions. Like other vertebrate calmodulins, lens calmodulin shows a calcium-dependent mobility shift on SDS-polyacrylamide gels and forms immune complexes with antiserum raised against vertebrate calmodulin. Lens calmodulin binds to the junction proteins MIP (main intrinsic protein, MW 26 Kdalton) and a 17.5 Kdalton polypeptide of lens fiber cells in a calcium-independent manner. Via calmodulin the junctions become calcium-sensitive.

Animals↗
PubMed1985

In vitro reassociation of EDTA-extractable proteins with calf lens fiber membranes.

Nature and site of membrane binding of the EDTA-extractable proteins (EEP) from calf lens fiber membranes have been studied. Reassociation of EEP to EEP-free lens fiber membranes only occurs by means of calcium, not by magnesium ions. This EEP-membrane binding is not hindered by the cytoskeleton. The proportional distribution of the EEP protein components is not altered by reattachment of EEP to the membrane. Calcium appears to be a limiting factor in the reassociation of EEP with the membrane. The total amount of reassociated EEP increases with increasing calcium concentration and may largely exceed the quantity of naturally occurring EEP in lens fiber membranes. In addition, the latter EEP-containing membranes are able to bind an additional amount of EEP in the presence of calcium. These results indicate that most of the EEP-binding sites in lens fiber membranes are not occupied by EEP. Trypsin- or Staphylococcus aureus V8 protease-treated fiber membranes retain the capacity to bind EEP in the presence of calcium. This result indicates that the small polypeptide fragment of the main intrinsic protein (MIP), which is accessible to proteolytic attack, very likely is not the membrane attachment point for EEP. It is suggested that phospholipids rather than membrane proteins are involved in the calcium-dependent binding of EEP to calf lens fiber membranes.

Animals↗
PubMed1985

Complex formation of EDTA-extractable proteins from calf lens fiber membranes with calcium and acidic phospholipids.

The nature of the membrane components, which are involved in the calcium-dependent binding of EDTA-extractable proteins (EEP) to calf lens fiber membranes, has been investigated. Association experiments of radioiodinated EEP with several lens membrane preparations by means of the gel-overlay technique have shown that only phospholipids have the ability to bind EEP in the presence of calcium. The water-soluble crystallins, cytoskeletal proteins and membrane proteins of lens cells lack EEP-binding properties in this technique. Binding studies of EEP with sonicated vesicles of separate phospholipids revealed that acidic calcium-binding phospholipids, i.e. phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) are effective in binding provided that calcium is present. Phosphatidylcholine (PC) and sphingomyelin (SM) do not bind EEP in the presence of calcium. The results of association and binding studies indicate that the EEP-lipid binding is purely electrostatic and is accomplished very likely by coupling of the anionic groups of EEP and phospholipids via calcium ions. The interaction of EEP with (isolated) lens fiber membranes is resistant to the non-ionic detergent Triton X-100 in the presence of calcium. This result confirms the earlier idea that the EEP-membrane binding is predominantly or even exclusively electrostatic. It is suggested that calcium-binding phospholipids, rather than proteins, function as binding sites for EEP in this hydrophilic calcium-dependent binding of EEP to lens fiber membranes.

Animals↗
PubMed1985

Biochemical and histochemical characteristics of proteins homologous to calf lens membrane proteins with high calcium-binding capacity.

Proteins with high affinity and capacity for calcium are present in the membranes of calf lens fiber and epithelial cells. They can be extracted from these membranes by means of EDTA or EGTA. The tissue specificity and localization of these 30-38 kD EDTA-extractable proteins (EEP) have been examined. Antibodies raised against calf lens fiber EEP specifically form immune complexes with distinct proteins of 30-38 kD in a great variety of non-lenticular tissues. By indirect immunofluorescence microscopy using anti-EEP antiserum, the EEP-like proteins could be detected in fibroblasts, retinal Müller cells, endothelial cells and some types of epithelial cells. Only covering epithelia (cornea, glomerulus) contained significant amounts of these proteins, irrespective of the shape of the cells. EEP-like proteins were absent in secreting epithelial cells of liver, kidney tubules and pancreas. In addition, they were not detected in muscle, nerve and fat cells, erythrocytes and lymphocytes. The localization and the number of EEP-like proteins varied among different cell types. In fibroblasts, containing only two EEP-like proteins (molecular weight (MW) 33.0 and 31.5 kD in calf tissue), predominantly the nucleus was stained. In vitro studies with permeabilized cultured fibroblasts from several species have shown that the nuclear staining was built up of bright spots around unstained nucleoli. In epithelial and endothelial cells of calf tissue, however, most fluorescent label was found in the plasma membranes. Immunoblotting experiments revealed the presence in these cell types of at least five EEP-like proteins, including a 33.0 and 31.5 kD component. The difference in staining pattern between these cells and fibroblasts might thus indicate that the nature or the localization of some of the EEP-like proteins is cell type-specific. Because of their extractability from various tissue membrane fractions by means of EDTA or EGTA it is suggested that at least part of the EEP-like proteins is bound to membrane structures via calcium. This characteristic feature, together with the MW values and the cross-reactivity with anti-EEP antiserum indicate that these proteins and the lens membrane proteins with high calcium-binding capacity share a very high degree of homology and may even be identical.

Animals↗
PubMed1985

Calcium-binding characteristics of the EDTA-extractable proteins from calf lens fiber membranes.

The calcium-binding characteristics of the EDTA-extractable proteins (EEP) from calf lens fiber membranes were studied by equilibrium dialysis and far-ultraviolet circular dichroism measurements. The EEP proteins appeared to contain binding sites with different affinities for calcium. These sites seem to behave as positively cooperating Ca2+ binding sites with a total capacity of 25 mol Ca2+ per mol EEP. The mean apparent dissociation constant (KD) for the Ca2+ binding sites was determined to be 7.7 microM. Calcium binding probably is accompanied by a decrease in the apparent alpha-helical content of the EEP proteins. The present results indicate that the EEP proteins belong to the group of proteins possessing high affinity and binding capacity for calcium. Because of the high calcium-binding capacity, the EEP proteins possibly function as an intracellular calcium store in the lens. The calcium-sensitivity of the conformational state of the EEP proteins, however, might point to a possible regulating function of these membrane proteins in calcium-dependent cellular processes in the lens.

Animals↗
PubMed1985

Bovine lens calmodulin. Isolation, partial characterization and calcium-independent binding to lens membrane proteins.

Calmodulin has been isolated from calf lens fiber cells. Like other vertebrate calmodulins lens calmodulin shows a calcium-dependent mobility shift on SDS-polyacrylamide gels and forms immune complexes with antiserum, raised against vertebrate calmodulin. Via the gel overlay technique radioiodinated calmodulin from lens or bovine brain was found to bind to the main intrinsic protein (MIP) and the 17.5 kDa protein of lens fiber membranes in a calcium-independent manner. After proteolytic digestion of lens fiber membranes with trypsin or Staphylococcus aureus V8 protease the calmodulin-binding activity of MIP is retained. This result indicates that the small polypeptide fragment of MIP, which is accessible to proteolytic attack, apparently is not the attachment point for calmodulin. Two additional calmodulin-binding proteins (MW 14 kDa and 16.5 kDa) are observed in junction-enriched fiber membrane fractions. These junction-specific proteins are bound to the membrane via calcium. In addition to MIP and the 17.5 kDa protein they are possibly involved in the calcium-dependent regulation of lens fiber junctions. The 14 and 16.5 kDa proteins are also present in epithelial membranes, prepared from freshly obtained calf lens epithelia. Whereas in the latter membranes the two proteins form part of the four 14-17 kDa major protein components, these proteins are absent in membranes from cultured lens epithelial cells. The epithelial 14 kDa and 16.5 kDa proteins thus appear to be junction-specific. The capacity of the latter proteins to bind calmodulin in the presence and absence of calcium indicates that these junction-specific proteins are very similar, if not identical, to the corresponding fiber junctional proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Immunological relationship between the EDTA-extractable proteins from calf lens fiber membranes.

An antiserum has been prepared against the EDTA-extractable proteins (EEP) from calf lens fiber membranes. It was shown to be highly specific for EEP. Using this anti-EEP antiserum in (crossed-line) immunoelectrophoresis and (crossed) immunoelectrofocusing experiments, evidence was obtained that all of the EDTA-extractable proteins are immunologically related. They have at least one of six different antigenic determinants in common, while some of the proteins with pI values above 4.8 probably have a second common determinant. The acidic proteins of EEP comprise specific determinants with a low immunogenicity. Furthermore, it was demonstrated that no crystallin-like determinant was present on the EEP molecules.

Animals↗
PubMed1983

Charge and molecular weight heterogeneity of EDTA-extractable proteins from calf lens membranes.

The EDTA-extractable proteins (EEP) of calf lens fiber cell membranes have been further characterized. Fiber EEP has been purified by gel filtration and resolved into eight bands with molecular weights of 30-38 K dalton by SDS-polyacrylamide gel electrophoresis. For epithelial EEP the same range has been obtained. In agreement with these findings a value of 33 K has been determined for fiber EEP by Sephadex G200 thin-layer gel filtration, while 34 K dalton was found by high-performance gel permeation chromatography in combination with low-angle laser light scattering (HPGPC-LALLS). The isoelectric focusing patterns of fiber and epithelial EEP show considerable charge heterogeneity. By two-dimensional electrophoresis the relation molecular weight-isoelectric point has been established for most EEP components. Peptide maps of the individual protein bands of fiber EEP differ from each other and from those of the beta Bp-, beta B1a- and beta B1b-crystallin bands, which have about the same molecular weight. From our results we conclude that EEP is not an oligomeric nor a multisubunit protein, but a collection of different extrinsic membrane proteins, biochemically unrelated to lens crystallins. The fact that removal of the cytoskeleton by urea-treatment of the membranes is a prerequisite for its isolation by EDTA or EGTA suggests that EEP is bound to the inner surface of the plasma membranes, probably via calcium.

Amino Acids↗