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Results for “Integrin alpha1beta1”

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PubMed2003

A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains.

A novel hemorrhagic metalloprotease, halysase, isolated from the snake venom of Gloydius halys induces apoptosis in endothelial cells. The purified metalloprotease is a monomeric glycoprotein with an isoelectric point of 4.8. Analysis of the cDNA sequence encoding halysase revealed that the enzyme consists of multifunctional domains including a proprotein domain, a protease domain, a disintegrin-like domain and a cysteine-rich domain. The metalloprotease has a DECD sequence in the disintegrin-like domain instead of the typical RGD sequence. Halysase strongly inhibits proliferation of human umbilical vein endothelial cells in a dose-dependent manner as well as adhesion of the cells to extracellular matrix proteins. The enzyme specifically hydrolyzes not only extracellular matrix proteins such as fibronectin, vitronectin, and type IV collagen, but also integrins alpha1beta1 and alpha5beta1. The apoptosis of endothelial cells induced by halysase is closely associated with activation of caspase-3 and decreased level of Bcl-X(L)/Bax. Apohalysase, which lacks metalloprotease activity, is also able to induce the apoptosis. Several lines of experimental evidence suggest that the protease domain and the disintegrin-like domain of halysase cooperatively contribute to the induction of endothelial cell apoptosis.

Amino Acid Sequence↗

PubMed1996

Platelet-derived growth factor and inflammatory cytokines have differential effects on the expression of integrins alpha 1 beta 1 and alpha 5 beta 1 by human dermal fibroblasts in vitro.

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet-derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4-day pig wounds were stained with antibodies specific for the alpha 1 and alpha 5 integrin subunits. Staining for alpha 1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for alpha 5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins alpha 1 beta 1, a collagen receptor, and alpha 5 beta 1 a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet-derived growth factor (PDGF)-AA, PDGF-AB, or PDGF BB. Each isoform of PDGF produced a significant decrease in the level of alpha 1 present on the cell surface and an increase in the level of alpha 5. Furthermore, PDGF-BB produced a corresponding decrease in alpha 1 mRNA and an increase in alpha 5 mRNA. In contrast, treatment with three inflammatory cytokines, IL-1 beta, TNF-alpha, and IFN-gamma, produced clear increases in the levels of alpha 1 and alpha 5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of alpha 1 and alpha 5 integrins by dermal fibroblasts during wound repair.

Animals↗

PubMed1995

Monocyte activation: rapid induction of alpha 1/beta 1 (VLA-1) integrin expression by lipopolysaccharide and interferon-gamma.

Monocytes play a key role in inflammation, tissue injury and remodelling and wound healing, and most monocyte effector functions are dependent on adhesive interactions. We have analyzed the changes in the pattern of beta 1 integrin expression that take place during monocyte activation and demonstrated that lipopolysaccharide (LPS) and interferon (IFN)-gamma specifically induce the expression of the alpha 1/beta 1 integrin, which was detectable on the monocyte membrane as early as 12 h after monocyte activation. The up-regulated alpha 1/beta 1 expression was not dependent on monocyte adherence to solid surfaces, and Northern blot analysis revealed that LPS and IFN-gamma induce the alpha 1 mRNA de novo. Monocyte deactivating cytokines such as interleukin (IL)-4 or IL-10, could only minimally inhibit the LPS- or IFN-gamma mediated up-regulation of alpha 1/beta 1, suggesting that cytokine release subsequent to monocyte activation does not play a major role in the integrin induction. Interestingly, the LPS-induced expression of alpha 1/beta 1 was found to be dependent on the redox state of the cell, since it was inhibited by antioxidants which also altered the morphological changes that take place during monocyte culture in vitro. The rapid induction of alpha 1 in LPS-activated monocytes suggests that alpha 1/beta 1 might be involved not only in monocyte/extracellular matrix interactions during inflammatory reactions, but also in contributing to further monocyte activation and cytokine production during septic shock syndrome.

Cell Adhesion↗

PubMed1996

Functional analysis of alpha 1 beta 1 integrin in human natural killer cells.

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

Animals↗

PubMed2005

beta1 integrins are not required for the maintenance of lymphocytes within intestinal epithelia.

beta(1) integrins are thought to play a central role in maintaining lymphocytes within mucosal epithelia via their interactions with extracellular matrix proteins and subepithelial cellular components within and underlying the basement membrane. In the current study type a (CD8alphabetaTCRalphabeta) and type b (CD8alphaalphaTCRgammadelta and CD8alphaalphaTCRalphabeta) intraepithelial lymphocyte (IEL) subsets within the mouse small intestine were found to express functional beta(1) integrin and the beta(1) integrin alpha chain partners alpha(1), alpha(2), and alpha(4). Using inducible beta(1) integrin-knockout bone marrow-chimeric mice we demonstrate that IEL expression of alpha(1) and alpha(2) but not alpha(4) is dependent on expression of the beta(1) chain. Importantly, deletion of the beta(1) chain in IEL did not alter the number or composition of lymphocytes within the intestinal epithelium. Thus, while IEL express functional beta(1) integrins, these are not required to maintain lymphocytes within intestinal epithelia. This result is discussed in the light of conventional views of intestinal lymphocyte homing and localization.

Animals↗

PubMed1995

Role of the alpha 1 beta 1 integrin complex in collagen gel contraction in vitro by fibroblasts.

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the beta 1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-beta, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the alpha 2 beta 1 and, to a lesser extent, the alpha 1 beta 1 integrin complexes. The present studies were undertaken to determine the role of the alpha 1 integrin chain, a collagen/laminin receptor, in collagen gel contraction by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the alpha 1 beta 1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the alpha 1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the alpha 1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the alpha 1 beta 1 integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.

Animals↗

PubMed1994

Expression and distribution of functional integrins in rat CNS glia.

In previous studies, we have reported on the expression of beta 1 integrins in type 1 astrocytes and their function in cell-substratum attachment (Tawil et al., J Cell Biol 120:261-271, 1993). Here we extend those findings by providing evidence that type 1 astrocytes express integrins of the beta 3 and possibly beta 4 subclasses and that the former (alpha v beta 3) functions in attachment by recognizing the peptide, Arg-Gly-Asp, in vitronectin. In addition, we have examined immunocytochemically the expression of beta 1 integrins on type 2 astrocytes and oligodendrocytes. The pattern of expression of integrins on these two cell types is distinct from type 1 astrocytes; most notably type 1 astrocytes but not oligodendrocytes or type 2 astrocytes express alpha 1 beta 1 heterodimers. Since type 2 astrocytes and oligodendrocytes originate from a common precursor (O-2A), the alpha 1 beta 1 heterodimer may be a functional marker which distinguishes O-2A-derived cells from those of the type 1 astrocyte lineage.

Amino Acid Sequence↗

PubMed1994

Loss of alpha 1 beta 1 and reduced expression of other beta 1 integrins and CAM in lung adenocarcinoma compared with pneumocytes.

Alterations in expression of various cell-adhesion molecules have been reported in a variety of malignant tissues. However, little is known about how lung adenocarcinomas differ in CAM expression from the normal lung. We analyzed the expression of integrins alpha 1 beta 1 through alpha 6 beta 1, intercellular adhesion molecule (ICAM)-1, neural cell adhesion molecule (NCAM), and lymphocyte function antigen (LFA)-3, CD44, and the two carbohydrate antigens, Lewisx (Le(x)) and sialosyl-Le-Le(x) of lung adenocarcinoma cells, and compared them with autologous pneumocytes. CAM expression was studied by an immunohistochemical method using monoclonal antibodies, and computerized image analysis was used to quantify the immunoperoxidase-staining intensity. The normal lung alveolar cells strongly expressed the integrins alpha 1 beta 1 and alpha 3 beta 1, and fairly expressed alpha 2 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1. ICAM-1, LFA-3, and CD44 were strongly expressed, whereas NCAM, the Le(x) and sialosyl-Le-Le(x) antigens, were expressed weakly. In contrast, we did not detect expression of the alpha 1 beta 1 integrin on any autologous lung adenocarcinoma cells, and they showed on average a 50% reduction in labeling relative intensity units for the integrin common chain marker beta 1, the specific integrins alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and ICAM-1, and LFA-3. Examination of the adjacent small blood vessel endothelium in malignant lung tissues did not reveal any major alterations in CAM expression, the small vessel endothelium of the normal and malignant lung tissues appeared with a similar CAM profile. These results suggest that lung adenocarcioma cells have a lack of alpha 1 beta 1 expression and significant reduction in some other integrin beta 1 and CAM expression in comparison with their autologous pneumocytes. This aberration in CAM expression by the lung adenocarcinoma cells may be involved in their loss of proliferation control and may interfere with leukocyte adhesion to tumor cells, enabling the tumor to escape immunodestruction.

Adenocarcinoma↗

PubMed1994

Anti-beta 1 integrin antibody inhibits Schwann cell myelination.

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypassed by adding a purified basal lamina component, laminin, to SC/neuron co-cultures. We have examined the role of laminin receptors, namely, the beta 1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the beta 1 subunit in association with the alpha 1 or alpha 6 subunit. In co-cultures of myelinating SCs, alpha 1 beta 1 is no longer present, alpha 6 beta 1 is still present but at reduced levels, and alpha 6 beta 4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that beta 1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of beta 1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-beta 1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an alpha 1 beta 1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the beta 1 subfamily of integrins other than alpha 1 beta 1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell.

Animals↗

PubMed1995

Antibody against human alpha 1 beta 1 integrin inhibits HeLa cell adhesion to laminin and to type I, IV, and V collagens.

In HeLa cells beta 1 integrin forms heterodimers with alpha 1, alpha 2, alpha 3, alpha 5 and alpha 6 integrin subunits. Integrin alpha v beta 5 can also be detected. A monoclonal antibody SR-84 identified the alpha 1 integrin subunit in immunoprecipitation assays and inhibited alpha 1-related cell adhesion to different matrix proteins, laminin-1 and type I, IV, and V collagens, whereas its effect on adhesion to type II collagen was marginal. HeLa cells do not attach to type VI collagen. The presence of magnesium was essential for HeLa cell adhesion, whereas calcium alone was not sufficient and high concentrations of calcium even counteracted the effect of magnesium. Cell adhesion to type I collagen was sensitive to changes in pH, unlike cell adhesion to type IV collagen. We conclude that SR-84 is a valuable tool to study alpha 1 integrin-related functions, and that in HeLa cells alpha 1 beta 1 integrin is a magnesium-dependent receptor for type I, IV, and V collagens but not for type II and VI collagens.

Antibodies, Monoclonal↗

PubMed1998

Regulation of cellular adhesion to extracellular matrix proteins by galectin-3.

The control of cellular adhesion to extracellular matrix proteins is poorly understood. In the present analyses, we set out to test the hypothesis that high galectin-3 concentration on the cell surface downregulates cellular adhesion to the extracellular matrix proteins. Various tumor cell lines were briefly incubated without or with galectin-3 and then allowed to adhere to wells coated with laminin-1, collagen IV and fibronectin. Our data demonstrated that the cells which were incubated with galectin-3 prior to plating had significantly reduced adhesion to extracellular matrix proteins. This inhibition involved the carbohydrate recognition domain of the lectin because adhesion was achieved in the presence of galectin-3 and lactose but not galectin-3 and sucrose. Furthermore we demonstrated that galectin-3 associates with alpha 1 beta 1 integrin in a lactose dependent manner.

Animals↗

PubMed2000

Reduced gut intraepithelial lymphocytes in VLA1 null mice.

Very late antigen 1 (VLA1) is an integrin collagen receptor that is expressed by lymphocytes in several disease states. VLA1 blockade has been shown to ameliorate gut disease in experimental graft-versus-host disease. Here we show that in the VLA1 null mouse, which is generally healthy, there is a 50% reduction in gut intraepithelial lymphocytes (IELs) despite an otherwise normal lymphocyte distribution in peripheral blood and lymphoid organs. The gammadelta to alphabeta ratios of IELs are unchanged. We also find that IL2-stimulated splenocytes from VLA1 null animals show a deficiency in adhesion to fibrillar and basement membrane collagen as well as reduced proliferation in response to collagen substratum. These results suggest that some, but not all, intraepithelial lymphocytes require VLA1 to survive or proliferate within the gut epithelium or possibly to traverse the basement membrane.

Animals↗

PubMed1994

Cell-collagen adhesion is inhibited by monoclonal antibody 33.4 against the rat alpha 1-integrin subunit.

A monoclonal antibody (mAb 33.4) is described which inhibits the adhesion and spreading of an adherent cell line (A-cells), established from Morris hepatoma 7777 and isolated hepatocytes on collagen IV but not on laminin, fibronectin, and vitronectin. mAb 33.4 retains its immunological activity after immobilization and covalent cross-linking to Protein G-Sepharose and is therefore a suitable tool for the preparative equimolar purification of two proteins with M(r) of 130 and 190 kDa from detergent-solubilized membrane fractions by immunoaffinity chromatography. The 190-kDa protein was identified as rat alpha 1-integrin subunit by N-terminal amino acid sequencing, while the 130-kDa protein was specifically stained by a beta 1-integrin subunit-specific antiserum. In immunoblot analysis mAb 33.4 recognized the 190-kDa band, suggesting that it is specific for the rat alpha 1-integrin subunit. The epitope recognized by mAb 33.4 is conformation-dependent because the staining in immunoblots was very strong if the SDS-PAGE was performed in the absence of reducing agents. The expression of alpha 1 beta 1-integrin in sinusoidal hepatocyte membrane domains of liver sections is shown by mAb 33.4 and antisera raised against the rat alpha 1- and beta 1-integrin subunits.

Animals↗

PubMed1995

Identification of integrins involved in cell adhesion to native and denatured type I collagens and the phenotypic transition of rabbit arterial smooth muscle cells.

Rabbit smooth muscle cells (SMC) in primary culture attached to and started proliferating on native and heat-denatured type I collagens, although the amount of cell attachment to denatured collagen was significantly lower. The cells adhered poorly and were unable to grow on commercial gelatin. In contrast, synthetic SMC in secondary culture could adhere to gelatin and grew as well on gelatin as on native type I collagen. The SMC in the contractile state adhered to native type I collagen through the alpha 1 beta 1 and alpha 3 beta 1 integrins. The cells in the intermediate phenotype also adhered to the substrate through the alpha 1 beta 1 and alpha 3 beta 1 integrins, but the relative amount of alpha 3 integrin decreased. The initial adhesion of cells in secondary culture to native type I collagen was mediated only by the alpha 1 beta 1 integrin. The cell-binding sequences did not contain DGEA (Asp-Gly-Glu-Ala) or RGD (Arg-Gly-Asp). In contrast, cell adhesion to heat-denatured type I collagen was mediated only by the alpha 1 beta 1 integrin in the contractile state and by the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 integrins in the synthetic state. In heat-denatured type I collagen, the sequences DGEA and RGD served as a recognition site for the alpha 2 beta 1 and alpha 3 beta 1 integrins. Our results suggest that rabbit SMC can recognize the native and denatured type I collagens through interactions with the triple helix-binding receptors and alpha chain-binding receptors and that the expression pattern of integrins changes in conjunction with the phenotypic properties of vascular SMC.

Amino Acid Sequence↗

PubMed1995

Establishment of a human hepatocyte line derived from primary culture in a collagen gel sandwich culture system.

A human hepatocyte line (HHY41) was established from normal human liver tissue. This cell line was derived from a primary culture of human hepatocytes maintained between two layers of collagen gel for 4 weeks. It differs from other human hepatocyte lines in that transfection with the simian virus 40 gene was not used for cellular transformation and nonhepatocellular coculture cells were not present. HHY41 cells have proliferated freely in serum and hormone-supplemented medium after more than 1 year in continuous culture, exhibiting typical morphological characteristics of hepatocytes. HHY41 cells retain glucose-6-phosphatase activity. They also retain the ability to secrete liver-specific proteins such as albumin, transferrin, and alpha-fetoprotein. Northern blot analysis confirmed the presence of albumin mRNA. Cytochromes P450 induced by polycyclic aromatic hydrocarbons are maintained in these cells. Detection of cell surface antigens revealed that HHY41 cells express alpha 1 beta 1-integrin, which is expressed by normal hepatocytes and not by bile duct epithelial cells. High-molecular-weight cytokeratin, a marker for bile duct cells, is also absent in HHY41. Cytogenetic analysis showed hyperdiploid karyotype with a consistent deletion in the short arm of chromosome 1. HHY41 can be considered a new human hepatocyte line which retains liver-specific functions of differentiated hepatocytes. Derived from normal liver tissue, not a hepatocellular carcinoma, it provides a new model system for studying the regulation of cell growth and differentiated functions in human hepatocytes.

Adult↗

PubMed2001

Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells.

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.

Animals↗

PubMed1995

Chemical cross-linking leads to two high molecular mass aggregates of rat alpha 1 beta 1 integrin differing in their conformation but not in their composition.

In order to detect protein interactions of the collagen/laminin receptor alpha 1 beta 1 integrin, covalent chemical cross-linking was performed with the homo-bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross-linking of the 190 kDa rat alpha 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M(r) 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of alpha 1 and beta 1 integrin subunits. No other cross-linked proteins associated with the alpha 1 or beta 1 subunit were detected. In contrast to the non-cross-linkable alpha 1 beta 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of alpha 1 beta 1 integrin to cross-link and produce two additional high molecular mass forms is shared by rat alpha 9 beta 1 integrin. Thus, the cross-linking approach directly indicates that beta 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.

Animals↗

PubMed1995

Characterization of molecular aggregates of alpha 1 beta 1-integrin and other rat liver membrane proteins by combination of size-exclusion chromatography and chemical cross-linking.

Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregates remains difficult; especially intermolecular complexes of membrane proteins tend to dissociate or artificially aggregate during detergent extraction out of membranes. Thus, the existence of protein aggregates was investigated by two approaches. First, after modest detergent extraction, the presence of three well characterized rat liver membrane proteins, alpha 1 beta 1-integrin, dipeptidyl aminopeptidase IV (DPP IV) and cell-CAM 105 (CAM = cell adhesion molecule), in aggregates could be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied detergents partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggregates. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpropionate) was combined with the performance of denaturating SEC. Under such optimized some high-molecular-mass complexes of all model proteins consisting of unknown components could also be detected. Taken together, non-denaturating SEC and chemical cross-linking in combination with denaturating SEC represent methodological approaches for the characterization of protein aggregates.

Adenosine Triphosphatases↗