cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations.

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.     

Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.     

Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase

We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).     

Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol

Two type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.     

Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.     

8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.

Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., D?skeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.     

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.     

Molecular architecture of a light-harvesting antenna. Core substructure in Synechococcus 6301 phycobilisomes: two new allophycocyanin and allophycocyanin B complexes

Two new allophycocyanin-containing complexes were found among the products of partial dissociation of the phycobilisomes of Synechococcus 6301 strain AN112. These complexes were purified to homogeneity and characterized with respect to composition, stability, and spectroscopic properties. The structures of the complexes were established to be (alpha AP beta AP)3 . 10.5K and (alpha 1APB alpha 2AP beta 3AP) . 10.5 K, where alpha AP and beta AP are subunits of allophycocyanin, and alpha APB is the subunit of allophycocyanin B (see Lundell, D. J., and Glazer, A. N. (1981) J. Biol. Chem. 256, 12600-12606), and 10.5K is an uncolored polypeptide of 10.5-kilodaltons. These complexes are derived from the core substructure of the phycobilisome. Electron microscopic studies of the morphology of the core of strain AN112 phycobilisomes (Yamanaka, G., Glazer, A. N., and Williams, R. C. (1980) J. Biol. Chem. 255, 11004-11010) as well as structural studies of an 18 S subassembly derived from the phycobilisomes by partial dissociation (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086) indicated that the core assembly consisted of two cylindrical elements each made up of the same four distinct "trimeric" biliprotein-containing complexes. Two such core components, (alpha AP beta AP)3 and alpha 2AP beta 2AP. 18.3K . 75K (where 18.3K and 75K are polypeptides of 18.3- and 75-kilodaltons), were shown to be contained within the 18 S subassembly (Lundell, D. J., and Glazer, A. N. (1983) J. Biol. Chem. 258, 894-901). The isolation of the two allophycocyanin-containing complexes described here completes the characterization of the four types of components in the Synechococcus 6301 phycobilisome core. Two lines of evidence indicate that each of the four complexes is present twice in the core: comparison of the compositions (and yields) of the complexes with that of the intact AN112 phycobilisome, and near-coincidence of the molar absorption spectrum of the phycobilisome with that generated by summing the spectra of the constituent complexes taken in appropriate molar proportions.     

Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes

Insulin-like growth factor (IGF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chem. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (beta-alpha-alpha-beta), apparent sizes of deglycosylated alpha (Mr = approximately 88,000) and beta (Mr = approximately 67,000) subunits, and amino acid composition of the subunits. Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric alpha beta form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the alpha 2 beta 2 form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their alpha subunits, which apparently show 60% identity. The IGF-I receptor alpha subunit lacks residues corresponding to the N-terminal 4 amino acids of the insulin receptor alpha subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.     

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.     

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B).

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.     

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.     

Structure of an ATPase operon of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The nucleotide sequence of the operon of the ATPase complex of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, has been determined. In addition to the three previously reported genes for the alpha, beta, and c (proteolipid) subunits of the ATPase complex (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1989) J. Biol. Chem. 264, 7119-7121), the operon contained three other genes encoding hydrophilic proteins with molecular masses 25, 13, and 7 kDa. The 25-kDa protein is the third largest subunit (gamma), the 13-kDa protein is most likely the fourth subunit (delta), and the 7-kDa protein may correspond to an unknown subunit of the ATPase, tentatively named as epsilon subunit. They do not have significant sequence similarity to subunits in F0F1-ATPases and eukaryotic V-type ATPases, whereas the other three subunits, alpha, beta, and c, have homologous counterparts in F0F1- and V-type ATPases. The order of the genes in the operon was delta alpha beta gamma epsilon c. The S. acidocaldarius ATPase operon differed from the eucabacterial F0F1-ATPase operon in that the former contains only one gene for a hydrophobic subunit at the most downstream part of the operon whereas the latter has three hydrophobic F0 genes preceding five hydrophilic F1 genes.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Beta-actinin is equivalent to Cap Z protein

Chicken skeletal muscle beta-actinin, previously reported to bind the slow-exchanging (pointed) ends of actin filaments was purified to homogeneity. By two dimensional gel electrophoresis, it consists of two subunits, beta I (35 kDa) and beta II (32 kDa), and each subunit has two isoforms. The amino acid sequences of V8 protease-digested peptides of beta I were nearly identical with those of portions of the muscle barbed end-blocking protein Cap Z alpha, although several amino acids were different from those deduced from cDNA sequences (Casella, J.F., Casella, S.J., Hollands, J.A., Caldwell, J.E., and Cooper, J.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5800-5804). The amino acid sequences of two peptides from beta II were completely identical with portions of Cap Z beta deduced from cDNA sequences (Caldwell, J.E., Waddle, J.A., Cooper, J.A., Hollands, J.A., Casella, S.J., and Casella, J.F. (1989) J. Biol. Chem. 264, 12648-12652). beta-Actinin capped the barbed end of an actin filament as evidenced by actin assembly of myosin S1-decorated filaments and specifically its impairment of growth in the "barbed" direction. Thus it is concluded that highly purified beta-actinin is identical with the more recently described Cap Z, an actin barbed-end capping protein of chicken skeletal muscle.