Intersubunit disulfides of the follitropin receptor

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.     

The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate)

The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.     

Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B

Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.     

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.     

Characterization of envelope proteins of alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 is a gammaherpesvirus which causes malignant catarrhal fever, an acute lymphoproliferative disorder of cattle and other susceptible Bovidae, which is almost invariably fatal. A preliminary analysis of proteins induced by the virus indicated that as many as six glycoproteins and one nonglycosylated molecule might be present in the virus envelope. Monoclonal antibodies selected for recognition of virion envelope proteins included two that recognized a complex of infected cell proteins, designated the gp115 complex, and neutralized virus infectivity in the absence of complement. The gp115 complex consisted of five glycoproteins of 115, 110, 105, 78, and 48 kilodaltons (kDa), and all except the 48-kDa species reacted with antibody in Western blots (immunoblots). Pulse-chase experiments analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions suggested that the 110-kDa protein was the precursor molecule which was processed by addition of sugars to 115 kDa. The 115-kDa protein was cleaved to form a disulfide-linked heterodimer of 78 and 48 kDa, which was the mature form of the molecule incorporated into the virion envelope. The glycoprotein contained N-linked sugars, but little or no O-linked sugar was present. The relative abundance of the mature protein and its ability to induce neutralizing antibodies suggest that it will prove useful to studies aimed at elucidating the biology and pathogenesis of alcelaphine herpesvirus 1.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

Cross-Linking Studies on the Membrane Topography of Photosystem I Reaction Center Complex in Synechococcus sp.

The subunit arrangement of the photosystem I reaction centercomplex in the thylakoid membranes of the thermophilic cyanobacteriumSynechococcus sp. was examined using three cross-linking reagents.(1) Treatments of osmotically shocked and NaBr-washed protoplastswith low concentrations of hydrophilic cross-linking reagents,dimethyladipimidate and glutaraldehyde, preferentially decreased62, 60, 14 and 13 kDa polypeptides of the photosystem I reactioncenter complex resolved by SDS-polyacrylamide gel electrophoresis,together with the anchor protein and allophycocyanin which areassociated with the outer surface of the thylakoid membranes.This suggests that these four subunits of the photosystem Icomplex are exposed on the stromal surface of thylakoid membranes.In contrast, a hydrophobic cross-linker, hexamethylenediisocyanate,unspecifically cross-linked most of the membrane polypeptides.(2) The 13 and 14 kDa polypeptides decreased always in parallelto each other on treatment of the protoplasts or isolatd CP1-awith the three cross-linking reagents, and the disappearanceof the two polypeptides was accompanied by the appearance ofa cross-linked product(s), when fixed with glutaraldehyde andhexamethylenediisocyanate. The results suggest that the 13 and14 kDa polypeptides are neighboring polypeptides in the complex. (Received June 7, 1986; Accepted November 13, 1986)     

Subunit composition of NDH-1 complexes of Synechocystis sp. PCC 6803: identification of two new ndh gene products with nuclear-encoded homologues in the chloroplast Ndh complex

Cyanobacteria contain several genes, annotated ndh, whose products show sequence similarities to subunits found in complex I (NADH:ubiquinone oxidoreductase) of eubacteria and mitochondria. However, it is still unclear whether the cyanobacterial ndh gene products actually form a single large protein complex or exist as smaller independent complexes. To address this, we have constructed a strain of Synechocystis sp. PCC 6803 in which the C terminus of the NdhJ subunit was fused to an His(6) tag to aid isolation. Three major NdhJ-containing complexes were resolved by blue native polyacrylamide gel electrophoresis, with approximate apparent molecular masses of 460, 330, and 110 kDa. N-terminal sequencing and mass spectrometry revealed that the 460-kDa complex contained ten annotated ndh gene products. Detergent-induced fragmentation experiments indicated that the 460-kDa complex was composed of hydrophobic (150 kDa) and hydrophilic (110-130 kDa) modules similar to that found in the minimal form of complex I found in Escherichia coli, except that the electron input module was not conserved. The difference in size between the 460- and 330-kDa complexes is attributed to differences in the stoichiometry of the hydrophilic and hydrophobic modules in the complex, either 2:1 or 1:1, respectively. We have also detected the presence of two new Ndh subunits (slr1623 and sll1262) that are unrelated to subunits in the eubacterial complex I but which have homologues in the closely related chloroplast Ndh complex of maize (Funk, E., Sch?fer, E., and Steinmüller, K. (1999) J. Plant Physiol. 154, 16-23). The presence of these additional subunits might reflect the use by the NDH-1 and Ndh complexes of a different, so far unidentified, electron input module.     

Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.     

Cell surface molecules that bind fibronectin's matrix assembly domain

The assembly of fibronectin into disulfide cross-linked extracellular matrices requires the interaction of mesenchymal cells with two distinct sites on fibronectin, the Arg-Gly-Asp cell adhesive site and an amino-terminal site contained within the first five type I homologous repeats (Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609). Proteolytically derived 29-kDa fragments of fibronectin (29kDa) containing these repeats bind to monolayers of cultured fibroblasts and inhibit fibronectin matrix assembly. The cell surface molecules interacting with fibronectin's 29-kDa matrix assembly domain have resisted purification using conventional methods such as affinity chromatography. Accordingly, in order to identify molecules which bind this fragment, 125I-labeled 29kDa was allowed to bind to fibroblast monolayers and chemically cross-linked to the cell surface with bis(sulfosuccinimidyl) suberate. Extraction of the cross-linked cell layer yielded radiolabeled complexes of 56, 150, and 280 kDa. Formation of these cross-linked complexes was specifically inhibited by the addition of excess unlabeled 29kDa but was unaffected by the presence of fibronectin fragments containing other type I repeats outside of the 29kDa matrix assembly domain. The cross-linked complexes were insoluble in nondenaturing detergents but soluble when denatured and reduced, suggesting that 29kDa may be cross-linked to components of the pericellular matrix. Immunoprecipitation of cross-linked cell extracts with a polyclonal antibody to fibronectin that does not recognize the amino terminus demonstrate that the 280-kDa band contains 29kDa cross-linked to fibronectin present on the cell surface. Formation of the 150-kDa complex was inhibited by EDTA, suggesting that divalent cations are required for its formation. Although the molecular mass and divalent cation requirement suggest that the 150-kDa complex may be related to an integrin, this complex was not immunoprecipitated by polyclonal antibodies generated to the alpha 5 beta 1 integrin fibronectin receptor.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues

Calcium/calmodulin-dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross-reactivity with affinity-purified polyclonal antibodies to the 50-kDa catalytic subunit of the brain calmodulin-dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One-dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two-dimensional fingerprints of the 125I-labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56-kDa and/or 58-kDa polypeptides of the skeletal muscle calmodulin-dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50-kDa and 60-kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two-dimensional peptide fingerprints, indicated considerable structural homology among the 50-kDa and 60-kDa subunits of the brain calmodulin-dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51-kDa subunit, skeletal muscle 56-kDa, and the brain 50-kDa and 60-kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin-dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3-5 mol phosphate/mol protein. The two-dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P-labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin-dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin-dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin-dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.     

Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle

Digitonin and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine-Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine-binding activity of 2500-2800 pmol/mg protein (60-70% purity) from digitonin-solubilized membranes by a combination of wheat-germ-agglutinin--Sepharose, anion-exchange and dihydropyridine-Sepharose chromatography steps. The individual subunits were isolated in dodecyl-sulfate-denatured form from the preparation of the receptor, enriched by a two-step large-scale procedure applied to Chapso-solubilized membranes. The 160-kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160-kDa subunit is cross-linked by intramolecular S-S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N-glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.     

Immunological characterization of rapeseed myrosinase

A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase-containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide-linked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.     

Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-beta superfamily

A bone-inductive protein has been purified from bovine bone and designated as osteogenic protein (OP). The purified OP induces new bone at less than 5 ng with half-maximal bone differentiation activity at about 20 ng/25 mg of matrix implant in a subcutaneous bone induction assay. The purified osteogenic protein is composed of disulfide-linked dimers that migrate on sodium dodecyl sulfate gels as a diffuse band with an apparent molecular weight of 30,000. Upon reduction, the dimers yield two subunits that migrate with molecular weights of 18,000 and 16,000. Both subunits are glycosylated. After chemical or enzymatic deglycosylation, the dimers migrate as a diffuse 27-kDa band that upon reduction yields two polypeptides that migrate at 16 kDa and 14 kDa, respectively. The carbohydrate moiety does not appear to be essential for biological activity since the deglycosylated proteins are capable of inducing bone formation in vivo. Amino acid sequences of peptides generated by proteolytic digestion show that the subunits are distinct but related members of the transforming growth factor-beta super-family. The 18-kDa subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16-kDa subunit is the protein product of the bovine equivalent of the human BMP-2A gene.     

Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.

The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.