A.L.M. de Leeuw

Fat-storing cells of the rat liver: Their isolation and purification

Abstract Fat-storing cells from the lobular area of the rat liver have been isolated by digesting the liver with pronase E and collagenase, and purified by Metrizamide density centrifugation and centrifugal elutriation. More than 70% of the cells in the final fraction were fat-storing cells. Per gram wet weight of liver, 3.1 ± 0.5 × 106 cells were isolated. The purified cells showed a well preserved ultrastructure and contained lipid droplets with a fluorescence characteristic of vitamin A. A HPLC technique demonstrated the presence of large quantities of retinol and retinyl palmitate in the isolated fat-storing cells.

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.

The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL

The small adaptor protein RIL consists of two segments, the C-terminal LIM and the N-terminal PDZ domain, which mediate multiple protein-protein interactions. The RIL LIM domain can interact with PDZ domains in the protein tyrosine phosphatase PTP-BL and with the PDZ domain of RIL itself. Here, we describe and characterise the interaction of the RIL PDZ domain with the zyxin-related protein TRIP6, a protein containing three C-terminal LIM domains. The second LIM domain in TRIP6 is sufficient for a strong interaction with RIL. A weaker interaction with the third LIM domain in TRIP6, including the proper C-terminus, is also evident. TRIP6 also interacts with the second out of five PDZ motifs in PTP-BL. For this interaction to occur both the third LIM domain and the proper C-terminus are necessary. RNA expression analysis revealed overlapping patterns of expression for TRIP6, RIL and PTP-BL, most notably in tissues of epithelial origin. Furthermore, in transfected epithelial cells TRIP6 can be co-precipitated with RIL and PTP-BL PDZ polypeptides, and a co-localisation of TRIP6 and RIL with Factin structures is evident. Taken together, PTP-BL, RIL and TRIP6 may function as components of multi-protein complexes at actin-based sub-cellular structures.

Lens membranes VI. Some characteristics of the EDTA-extractable protein (EEP) from bovine lens fiber membranes

Abstract After urea-treatment of calf lens fiber membranes, a well defined extrinsic membrane protein fraction (EEP) can be extracted by EDTA-solution. It contains polypeptides of mol. wt. 32K and 35K (SDS-gel electrophoresis). Thin-layer gel filtration shows that without detergent no oligomers are formed. It is easily lost by its adsorption characteristics, however, it can be purified from contaminating α crystallin by gel filtration. The 35K fraction comprises components with isoelectric points at 4·6–4·7 (double band) and between 6·2 and 7·2. The 32K fraction shows components at 4·6–4·7 and 6·2 as was found by two-dimensional gel electrophoresis. The method of isolation, the isoelectric focusing pattern, and the amino acid and mol. wt. composition distinguish EEP clearly from the soluble crystallins. EEP comprises two distinct antigenic components with isoelectric points between 6·2 and 7·2. This was revealed by immunoelectrofocusing of EEP vs. anti-EEP antiserum from rabbit. Combining the results of two-dimensional electrophoresis and immunoelectrofocusing it was found that the 32K and 35K polypeptide have different antigenic properties. Traces of crystallins are difficult to remove. However, by means of specific anti-crystallin antisera it has been shown that EEP has a determinant in common with γ crystallin.

Lens membranes. XII. Age-related changes in polypeptide composition of bovine lens fiber membranes

Changes in the polypeptide composition of urea-treated bovine lens fiber membranes in relation to age have been studied. By means of SDS-gel electrophoresis it has been shown that the relative amount of EDTA-extractable extrinsic membrane protein (EEP) as compared to the amount of 23 K dalton plus 26 K dalton band protein (MIP) decreases with age. Simultaneously, the 26 K dalton protein is converted into the 23 K dalton protein. Both MIP and the 23 K dalton band protein are chloroform-methanol extractable and show heat induced aggregation by boiling in sodium dodecyl sulfate solution. We propose that the 23 K dalton protein is a degradation product of MIP. Limited trypsin treatment of the membrane yields a polypeptide with an apparent molecular weight of 22 K dalton originating from MIP, suggesting a cleavage similar to those in vivo. Some characteristics of MIP are similar to those of the major mouse hepatic gap junction protein.

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.

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)

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.

Lens membranes XIII. Comparative study of biochemical characteristics of the fiber membrane polypeptides from bovine, pig, sheep and chicken lenses

The polypeptides of the lens fiber membranes of bovine, pig, sheep and chicken lens have been compared by means of SDS-gel electrophoresis. The major polypeptides of the lens membranes of the mammalian species are: the main intrinsic protein (MIP, 26 000 dalton), the EDTA-extractable protein (EEP, 35 000 and 32 000 dalton) and band I protein (17 000–20 000 dalton). Limited trypsin treatment converts MIP to a 22 000 dalton protein. Limited proteolysis with protease from Staphylococcus aureus V8 results in stepwise breakdown of MIP to a 23 500 and 22 500 dalton protein. Boiling in sodium dodecyl sulfate solution induces heat aggregation of the mammalian MIP and its 23 000 dalton degradation product, which is formed during ageing. These two polypeptides can be extracted from the fiber membranes by chloroform-methanol. EEP can be extracted from the membranes of each species by EDTA-solution. The mammalian EEP preparations have similar polypeptide compositions. However, the isoelectric focusing patterns of calf and sheep EEP have more bands in common than those of calf and pig. The major polypeptides of chicken lens membranes are: MIP (27 000 dalton), EEP (35 000 and 32 000 dalton) and a 44 000 dalton protein. Limited treatment of these membranes with trypsin or protease ( Staphylococcus aureus V8) results in conversion of MIP to a 22 000 or a 22 500 dalton polypeptide, respectively. Chicken lens MIP is not readily extracted by chloroform-methanol and does not aggregate by boiling in SDS, in contrast to MIP from mammalian lens. Extraction with EDTA results in an EEP preparation which comprises, in addition to the 32 000 and the 35 000 dalton polypeptides, a 68 000 dalton polypeptide. The isoelectric focusing pattern of chicken EEP differs clearly from that of the mammalian EEP preparations, while the overall isoelectric focusing patterns of the four investigated EEPs are greatly different from those of the corresponding lens crystallins.

Calcium-dependent binding of EDTA-extractable proteins to calf lens fiber membrane structures.

Calf lens fiber membranes and fractions enriched in junction-like structures have been isolated in the absence and presence of EDTA. Their biochemical features have been studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting experiments have provided evidence that a distinct group of EDTA-extractable proteins, being one of the main protein components of calf lens fiber membranes and very likely also of junction-like structures, is bound to these membranes via calcium ions. In addition to these proteins, four polypeptides with apparent molecular weights between 14000 and 17000 are characteristic for detergent-insoluble lens fiber structures prepared in calcium-rich medium. The absence of EDTA-extractable proteins in the urea-soluble calcium-containing fraction implies that they are not components of the cytoskeleton and that the calcium-dependent binding of these proteins to the membrane is urea-resistant. The use of EDTA throughout the whole membrane isolation procedure results in their complete removal from the membranes which already starts during buffer washing. This indicates that EDTA-extractable proteins exclusively consist of extrinsic membrane proteins which probably are not involved in cytoskeleton binding.