Differences In Apparent Molecular Weight Research Articles

Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported DisclosuresNo relevant conflicts of interest to declare.

Structural Characterization of the Fibroblast Growth Factor-binding Protein Purified from Bovine Prepartum Mammary Gland Secretion

A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.

Charge and molecular weight differences for fourteen guaiacol peroxidase isozymes in flax indicate that most are encoded by different structural genes.

Ferguson plots, linear regressions of log(relative mobility) against polyacrylamide gel concentration, provide an electrophoretic method of simultaneously obtaining estimates of net charge and molecular weight for nondenatured proteins, and are a powerful method of accurately comparing these characteristics among isozymes within an enzyme system. Here, Ferguson plots compare the characteristics of the nine isozymes (three cationic and six anionic) usually seen in soluble extracts of flax (Linum usitatissimum) seedling tissues, and of five anionic isozymes induced by heavy metal stress. Despite some differences in apparent molecular weight, all but one of the isozymes separated from each other on the basis of net charge and, except for a second isozyme with a comparatively high molecular weight, the net charges of the isozymes ranked in order of their relative mobilities on nondenaturing gels. Differences among the isozymes make it unlikely that any are related, which indicates that they are encoded by different structural genes.

Cholinesterases from the common oyster ( Crassostrea gigas) : Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors

Marine bivalves such as oysters and mussels are widely used as bioindicators of contamination in the monitoring of pollutant effects. As filter feeders, these species are known to be good general indicators of chemical contamination. However, the efficient use of decreased acetylcholinesterase activity in the oyster as a biomarker of exposure to neurotoxic compounds requires a definition of the different types of cholinesterases coexisting in this mollusk. This study reports the partial purification, separation and characterization of two cholinesterases extracted from the oyster Crassostrea gigas. Differences in apparent molecular weight, type of glycosylation and hydrophobicity, and sensitivity to inhibitors suggest that they are encoded by two different genes. `A' cholinesterase (apparent molecular weight 200 kDa) is anchored to the membrane via a glycolipid, is not glycosylated but sensitive to organophosphate and carbamate inhibitors. `B' cholinesterase (molecular weight 330 kDa) is hydrophilic, glycosylated and highly resistant to organophosphate and carbamate inhibitors. The kinetic properties of these two cholinesterases were compared with those of other invertebrate cholinesterases. The presence of a cholinesterase insensitive to insecticides suggests that a significant improvement in the use of oyster cholinesterases as biomarkers of pollutant effects could be achieved by simple separation of the two forms.

A Comparison of the Renal and Hepatic Microsomal Glucose-6-phosphatase Enzymes

The liver glucose-6-phosphatase enzyme has been extensively characterized and relatively little is known about the renal microsomal glucose-6-phosphatase enzyme. The reason for lack of study of the renal glucose-6-phosphatase enzyme is that it has been assumed to be the same as the liver enzyme. Immunoblotting with antibodies raised against the liver enzyme revealed differences in apparent molecular weight and antigenicity between the liver and kidney glucose-6-phosphatase enzyme proteins. Characterization of the activity of the renal glucose-6-phosphatase enzyme also showed that it is regulated differently to the liver enzyme in some metabolic states. This implies that the renal and liver glucose-6-phosphatase enzymes may have different roles.

Phosphorylation of the Transcription Factor NFATp Inhibits Its DNA Binding Activity in Cyclosporin A-treated Human B and T Cells

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.

Cloning of the cDNA Encoding Human Platelet CD36: Comparison to PCR Amplified Fragments of Monocyte, Endothelial and HEL Cells

Glycoprotein CD36, also known as GPIIIb or GPIV, is a major platelet glycoprotein that bears the newly identified Naka alloantigen. The aim of this study was to clone platelet CD36 and investigate other forms of CD36-cDNA present in monocytes, endothelial and HEL cells. RNA from above mentioned cells were reverse transcribed (RT), using specific primers for CD36, and amplified by the polymerase chain reaction (PCR) technique. Sequencing the different amplified platelet derived cDNA fragments, spanning the whole coding and flanking regions, showed the near identity between platelet and CD36-placenta cDNA. Platelet CD36-cDNA cross-hybridized, in Southern blots, with RT-PCR amplified cDNA originating from monocytes, endothelial and HEL cells. However, monocytes showed a RT-PCR amplified cDNA fragment (561 bp) that was present in platelets and placenta but not on endothelial on HEL-cells. Northern blot analysis of platelet RNA hybridized with placenta CD36 indicated the presence of a major (1.95 kb) and a minor (0.95 kb) transcript. The 1.95 kb transcript was the only one observed on Northern blots of monocytes, endothelial and HEL cells. These results indicate that the structure of CD36 expressed in platelets is similar, with the exception of the 3' flanking region, to that of placenta. Differences in apparent molecular weight between CD36 and CD36-like glycoproteins may be due to post-translational modifications.

Structural and functional impairment of surfactant protein A after exposure to nitrogen dioxide in rats.

The major surfactant protein A (SP-A) determines the dynamics of surfactant metabolism and function. We now for the first time studied the in vivo exposure of the SP-A to nitrogen dioxide (NO2) and compared it to in vitro exposure effects. Several properties that are believed to possess physiological functions were investigated. Exposure of rat SP-A to NO2 showed 1) a higher alveolar pool size after in vivo exposure, 2) a slightly decreased mannose binding capacity for the in vivo exposed form but a highly decreased binding after in vitro exposure, 3) an affected protein-lipid aggregation that was impaired most by the in vitro exposed protein, 4) a low inhibition for the secretion of phosphatidylcholine in isolated type II pneumocytes, and 5) no difference in apparent molecular weight and immunological reactivity of the differently exposed SP-A. The NO2-induced alterations of the SP-A may contribute to the pulmonary toxicity of this oxidant. It is clear from this study that the extend of the effects from in vivo and in vitro exposure are different. Therefore both forms of exposure are necessary to assess the biological significance of NO2 exposure.

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87–116 kDa, 38–44 kDa and 26–28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87–116 kDa and 38–44 kDa.

Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene

Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.

Clonal analysis of non-typable Haemophilus influenzae by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole cell polypeptides

A collection of 223 strains of non-typable Haemophilus influenzae was assembled. Most strains were isolated from hospital in-patients in North East Scotland between 1984 and 1989. These isolates were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell polypeptides. Variability was assessed in terms of apparent molecular weight differences between the protein profiles. Isolates were grouped on the basis of Dice coefficients of similarity and assigned to clones. Of the 223 strains, 147 were unique; the remaining strains were assigned to multi-member clones of which 13 clones had two members, one clone had three members, three clones had four members, three clones had five members, two clones had six members and one clone had eight members. The longest time interval between isolation of clonally related strains virtually equalled the limits of the study. Isolates from a childrens' hospital were significantly less diverse than those from patients in adult wards.

The hemagglutinating adhesin HA-Ag2 of Bacteroides gingivalis is distinct from fimbrilin.

We carried out a series of immunoblots with antigenic preparations from the periodontal pathogen Bacteroides gingivalis using antisera of restricted specificity for the hemagglutinating adhesin HA-Ag2, and for the major structural subunit of the fimbriae (fimbrilin). We have been able to show that these 2 antigens are distinct. The fimbrilin subunit had an apparent molecular weight of 42 kDa in all of the bacterial preparations tested. HA-Ag2 occurred as a pair of bands at 43 and 49 kDa in outer membranes prepared as extracellular vesicles, and at 33 and 38 kDa in glass-bead-EDTA extracted antigens and in sheared-cell outer membranes prepared in the presence of EDTA. No HA-Ag2 was found in an enriched fimbrial preparation. The 2 antigens could thus be distinguished on the basis of their behaviour when subjected to different extraction techniques. The lower apparent molecular weight of HA-Ag2 (a pair of bands at 33 and 38 kDa) was invariably associated with the presence of EDTA in the buffers used to prepare the extracts, and the effect could be partially prevented by adding MgCl2 to the extraction buffer. The difference in apparent molecular weight of HA-Ag2 in the different extracts can thus be attributed either to an EDTA-sensitive tertiary conformation of its component polypeptides, or to an EDTA-sensitive linkage of each of these polypeptides to an unknown component of approximately 10 kDa.

Modulation of extracellular matrix proteins in rat liver during development

The expression and localization of extracellular matrix proteins in rat liver was investigated as a function of liver development. Levels of extracellular matrix proteins were measured by dot-blot or immunoblot protocols using monospecific antibodies prepared against collagen types I, III and IV; laminin; fibronectin; and fibronectin receptor. Proline and hydroxyproline levels from extracted liver collagen were quantitated by Pico Tag analysis. It was observed that the content of type IV collagen and fibronectin in the rat liver increased two to four times during the perinatal period. In contrast, levels of laminin and collagen types I and III decreased up to twofold in developing rat livers. The content of fibronectin receptor during ontogeny was decreased four times in an inverse relationship to fibronectin molecules. Fibronectin receptor and extracellular matrix proteins displayed no difference in apparent molecular weight as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots. Indirect immunofluorescence staining of frozen thin liver sections revealed that the pattern of localization of extracellular matrix proteins in the nonvascular regions of fetal liver was punctate rather than restricted to a specific region such as the perisinusoidal area of adult livers. Similarly, fibronectin receptor was also present, mainly in the sinusoidal area of adult livers, whereas fetal sections were diffusely stained. Our findings suggest that the differential modulation of extracellular matrix proteins and their localization in the developing rat livers undergo a dramatic alteration in the composition and structural organization of matrix material, which may act to modulate proliferation and to promote the differentiation of liver cells during development.

Biochemical Characterization of the η Chain of the T-cell Receptor: A Unique Subunit Related to ζ

Abstract The T-cell antigen receptor is a multisubunit complex consisting of at least seven chains. Based upon structural and genetic considerations, we have divided these chains into three groups. The alpha and beta subunits (Ti) are the clonotypic chains responsible for antigen recognition. Three chains that are invariant among all T-cells define the CD3 complex. These include the CD3 gamma, delta, and epsilon chains. The zeta chain is a distinct component that, like the CD3 chains, is invariant among all T-cells. In the majority of receptors, zeta is found as a disulfide-linked homodimer. We have recently shown that approximately 10% of zeta is disulfide-linked to a chain which we have called eta. A preliminary model has been proposed, suggesting that there are two subclasses of receptors, depending upon the presence within the complex of either the zeta-zeta homodimer or the zeta-eta heterodimer. Evidence has been presented that these two subclasses may perform distinct signaling functions. In this paper the eta chain is characterized to determine whether it is structurally related to the zeta chain and, in particular, whether it might represent a post-translational modification of zeta. We can identify specific antigenic epitopes that are shared by both zeta and eta. However, not all antibodies raised against zeta can directly recognize eta. The apparent molecular mass of eta is 22 kDa, whereas zeta has a molecular mass of 16 kDa. We are unable to demonstrate any post-translational covalent modifications of eta to explain the difference in apparent molecular weight. These include phosphorylation, glycosylation, or sulfation. Amino acid incorporation studies demonstrate that the amino acid composition of eta is distinct from that of zeta. All of the eta in a T-cell is found in association with the rest of the components of the T-cell receptor. In addition, our anti-eta antibodies allow us to directly recognize human eta, which has an apparent molecular mass of approximately 23 kDa. Thus, eta and zeta appear to be related but distinct proteins, and we would propose that eta is the second member of the zeta group of components of the T-cell receptor.

Folate coenzyme and antifolate transport proteins in normal and neoplastic cells

The transport systems for folate coenzymes and antifolate compounds into various types of normal and neoplastic cells display considerable diversity in their pharmacokinetics and also in terms of the apparent molecular weights of the proteins involved. Further, several uptake routes may exist in a given cell type. A variety of neoplastic tissues have been reported to rely upon a single major transport system that has a relatively high affinity for the reduced form of folate compounds and for antifolates such as methotrexate. Using a photoaffinity analogue of methotrexate, we have identified the involvement of a 48 kDa membrane protein and a 38 kDa cytosolic or peripheral membrane protein in the transport of this compound into murine L1210 leukemia cells. Such an uptake is absent in mutant L1210 cells that are defective in methotrexate transport. We propose a model for the uptake of reduced folate coenzymes in L1210 cells in which the compound is initially transported across the cell membrane by the 48 kDa protein and delivered on the cytoplasmic surface to the 38 kDa protein; the 38 kDa protein then carries the folate compound to a specific enzyme of folate metabolism. Antibodies to the membrane folate binding protein from human placenta cross-react with the 48 kDa protein in L1210 cell membranes indicating an immunological relationship between these two proteins. Comparison of the amino acid sequences of peptides of the placental receptor obtained by digestion with S. aureus V8 protease indicate the presence of two homologous forms of the folate binding protein in placenta; one of these forms appears to have an identical sequence to the soluble and membrane associated folate binding proteins in human epidermoid carcinoma (KB) cells, which in turn share the primary structure of the soluble and membrane associated folate binders in human milk in the regions that have been sequenced. These results indicate that the folate coenzyme transport proteins in various tissues may be structurally related and in several instances may even be identical. In the latter cases the observed differences in apparent molecular weights may be due to differences in glycosylation and/or proteolysis. In support of this view is our observation that the deglycosylated and/or partially proteolyzed placental receptor retains the ability to bind folate.

Malate dehydrogenase isozymes in flax genotroph leaves: differences in apparent molecular weight and charge between and within L and S.

Ferguson plots demonstrated that corresponding malate dehydrogenase (MDH) isozymes of Durrant's L and S flax genotrophs differ in apparent molecular weight (MW) and also in net negative charge. The MW differences explain heritable differences in electrophoretic relative mobility (Rm) between corresponding L and S isozymes. The MW for each MDH isozyme was higher for L than for S and resulted in a slower Rm for L. The net negative charge for each isozyme was higher for L than for S. MDH isozymes also differ in MW within L and S. MW was lower for isozymes in leaves from the bottom of the stem than in leaves from the top of the stem, particularly in L. Integration of information on the MDH isozyme system in the flax genotrophs and information on the peroxidase system suggests the possibility that common modifier loci may control Rm in both enzymes.

Rat liver insulin mediator which stimulates pyruvate dehydrogenase phosphatase contains galactosamine and D-chiroinositol

It has been established that insulin treatment of cells, isolated plasma membranes, or whole animals leads to the generation of low molecular weight mediators which serve as intermediates in the signalling pathway. At least two distinct classes of mediator have been described, based on differences in apparent molecular weight, isoelectric point and biological activity (Cheng, K., and Larner, J. (1985) Ann. Rev. Physiol. 45, 407-424). Recently, Saltiel's (Saltiel, A.R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5793-5797) and Mato's (Mato, J.M., Kelly, K.L., Abler, A., and Jarett, L. (1987) J. Biol. Chem. 262, 2131-2137) laboratories have described an insulin "modulator" which was apparently derived from glycosylphosphoinositol linker, similar to those known to anchor proteins to the external surface of the cell membrane (Low, M.G. (1987) Bioch. J. 244, 1-13). In this paper, we report that highly purified preparations of the insulin mediator which stimulates pyruvate dehydrogenase phosphatase contain mannose, galactosamine, and D-chiroinositol. These determinations are based upon analyses using paper chromatography and gas chromatography/mass spectroscopy. Nitrous acid deamination of the mediator resulted in release of inositol phosphate, indicating that the galactosamine and D-chiroinositol are linked. Although the presence of chiroinositol in modulator from H35 hepatoma cells has been recently reported (Mato, J.M., Kelly, K.L., Abler, A., Jarett, L., Corkey, B.E., Cashel, J.A., and Zopf, D. (1987) Bioch. Biophys. Res. Comm. 146, 764-770), the optical identity of the inositol remained unknown until the present report. Likewise, the presence of galactosamine rather than glucosamine in insulin mediator is a novel finding. These findings, coupled with those of Saltiel and Mato's groups, provide clear evidence for the existence of multiple forms of insulin mediators. Additionally, the results presented here afford further confirmation for the formation of insulin mediators from glycosyl-phosphoinositol linkers.

Modification of the apparent molecular weight of different benzodiazepine binding proteins from rat brain membranes by various endoglycosidases.

Membranes from rat cerebellum or hippocampus were photolabeled by [3H]flunitrazepam and incubated with various endoglycosidases. The largest reduction in apparent molecular weight of photolabeled proteins P51 (mol. wt. 51,000) and P55 (mol. wt. 55,000) was obtained using endoglycosidase F or glycopeptidase F. However, even after extensive treatment with these enzymes the deglycosylated products of proteins P51 (apparent mol. wt. 44,000) and P55 (two peptides with apparent mol. wt. 48,000 and 51,000) still differed in their apparent molecular weight. This might indicate that structural features other than N-linked carbohydrates are responsible for the difference in apparent molecular weight of these proteins.

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain.

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

An Integral Membrane Protein Antigen Associated with the Membrane Attachment Sites of Actin Microfilaments is Identified as an Integrin β-Chain

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.