Beta Complex Research Articles

Cytokine and inflammation: role of IL-5 and its receptor system in inflammation

The inflammatory response is mediated by various cytokines. IL-5 is one of the inflammatory cytokines. The study of IL-5 has its origins in the search for one of the B-cell differentiation factors, named T-cell-replacing factor (TRF), that induces antigen-stimulated B cells to differentiate into antibody-forming cells. Eosinophil-differentiation factor, EDF, is a factor produced by thoracic duct lymphocytes of parasite-infected rats. cDNA cloning and mAb against IL-5 enable us to identify this molecule as a cytokine (TRF/EDF) that has pleiotropic activity on various target cells besides B cells and eosinophils. The pleiotropic activity of IL-5 is directly dependent on the initial binding to the IL-5 receptor (IL-5R) on the target cell surface. IL-5 transduces its signals through high affinity IL-5R which is constructed by two distinct polypeptides, alpha and beta. IL-5R alpha binds IL-5 with low affinity and associates with the beta chain which can convert low affinity IL-5R to high affinity IL-5R. IL-5R beta that does not bind IL-5 by itself is essential for the IL-5 signaling and is shared among IL-5R, IL-3R, and GM-CSFR. These results imply why IL-5, IL-3, and GM-CSF are eosinophylopoietin. The truncated IL-5R alpha and IL-5R beta complexes can not transduce IL-5 signals, although they bound IL-5 with high affinity, suggesting that IL-5-specific signaling may be transduced through IL-5R alpha.

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.

The alpha beta complexes of ATP synthase: the alpha 3 beta 3 oligomer and alpha 1 beta 1 protomer.

The basic structures of the catalytic portion (F1, alpha 3 beta 3 gamma delta epsilon) of ATP synthase are the alpha 3 beta 3 hexamer (oligomer with cooperativity) and alpha 1 beta 1 heterodimer (protomer). These were reconstituted from the alpha and beta subunits of thermophilic F1 (TF1), and the alpha 3 beta 3 hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F1 and TF1, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.

Type-Specific Regulation of Adenylyl Cyclase by G Protein βγ Subunits

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) dissociate into guanosine triphosphate (GTP)-bound alpha subunits and a complex of beta and gamma subunits after interaction with receptors. The GTP-alpha subunit complex activates appropriate effectors, such as adenylyl cyclase, retinal phosphodiesterase, phospholipase C, and ion channels. G protein beta gamma subunits have been found to have regulatory effects on certain types of adenylyl cyclase. In the presence of Gs alpha, the alpha subunit of the G protein that activates adenylyl cyclase, one form of adenylyl cyclase was inhibited by beta gamma, some forms were activated by beta gamma, and some forms were not affected by beta gamma. These interactions suggest mechanisms for communication between distinct signal-transducing pathways.

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.

A major substrate of maturation promoting factor identified as elongation factor 1 beta gamma delta in Xenopus laevis

Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Bellé, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.

Organization and nucleotide sequence of the rat T cell receptor beta-chain complex.

We have characterized four overlapping genomic clones containing the DA rat TCR C beta complex, which span a total of 23 kb and bear two closely related complexes of gene segments. The D beta 1-J beta 1-C beta 1 and the D beta 2-J beta 2-C beta 2 complexes each contain a single diversity segment, six joining segments and four exons that encode the C region. All gene segments appear to be functional except J beta 2.5, which has a 5-bp frame-shifting deletion. This organizational pattern is identical to that of the mouse, and the homologous rat and mouse coding regions share about 92% nucleotide sequence identity. Our sequence comparisons indicate that a localized gene correction event has homogenized the sequences of the first exons of C beta 1 and C beta 2 in the evolutionary time since rats and mice became separate species. We have identified three repetitive elements, each flanked by short direct repeats, present in the region "brain-specific" identifier (ID) sequences, another is a truncated member of the LINE I class of repetitive elements, and the third is a member of the Alu type 2 family. The insertion of at least two, and probably all, of these elements has occurred since the time of rat/mouse divergence. We have identified a substantial number of "cryptic" rearrangement signals (heptamer/nonamer) in the C beta locus, which match the consensus sequence as well or better than authentic signals, and may represent sites of nonfunctional rearrangements.

Interaction of the IL-2 Receptor with the src -Family Kinase p56 lck : Identification of Novel Intermolecular Association

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.

Latent Forms of Transforming Growth Factor-β (TGFβ) Derived from Bone Cultures: Identification of a Naturally Occurring 100-kDa Complex with Similarity to Recombinant Latent TGFβ

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.

A soluble factor released by CD8+CD57+ lymphocytes from bone marrow transplanted patients inhibits cell-mediated cytolysis

We report a new inhibitory activity of CD8+CD57+ peripheral blood lymphocytes from allo-bone marrow transplant patients. Positively selected CD8+CD57+ lymphocytes act as potent inhibitors of allospecific cytolytic T lymphocyte and lymphokine activated killer cell cytolytic activities. These suppressor cells are mature T cells expressing the CD2, 5, 7, CD3-TcR alpha beta complex, and lack natural killer cell markers as well as cytolytic function. Their inhibitory activity on both cytolytic processes and T-cell proliferation is mediated by a non- antigen-specific soluble factor released by CD8+CD57+ cells in culture supernatants. Preliminary characterization suggests that the CD8+CD57+ cells' inhibitory activity is mediated by a low molecular weight, glycosylated factor as indicated by its less than 1S coefficient of sedimentation and its binding to concanavalin A lectin.

Effects of non-MHC loci on resistance to retrovirus-induced immunodeficiency in mice.

Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1n and H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1b and other H-2 haplotypes including b, s, and q. The Fv-1b, H-2r strain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII x RIIIS) F1, F2 and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V beta complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.

G-protein .beta..gamma. forms: identity of .beta. and diversity of .gamma. subunits

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Extraction and purification of the beta subunit and an active alpha beta-core complex from the spinach chloroplast CFoF1-ATP synthase.

Incubation of Rhodospirillum rubrum chromatophores with 2 M LiCl in the presence of MgATP has been shown to remove their F1 beta subunit leaving inactive but fully reconstitutable beta-less chromatophores (Gromet-Elhanan, Z., and Khanashvili, D., (1986) Methods Enzymol, 126, 528-538). A similar treatment of thoroughly washed spinach thylakoids has now been shown to release the CF1 beta subunit (CF1 beta) together with a complex containing equal amounts of CF1 alpha and CF1 beta (CF1 (alpha beta]. The purified CF1 (alpha beta) complex can reconstitute an active membrane-bound hybrid F0F1-ATPase with beta-less R. rubrum chromatophores and also catalyzes a low but very reproducible soluble MgATPase. Purified CF1 beta shows none of these activities although it can bind as efficiently as CF1 (alpha beta) to the beta-less chromatophores. By subjecting the crude spinach 2 m LiCl extract to dissociating conditions an enriched CF1 beta preparation is released. It contains traces of CF1 alpha and CF1 delta, is able to reconstitute an active hybrid F0F1-ATPase but, as the pure CF1 beta shows no soluble ATPase activity. These results indicate that trace amounts of CF1 alpha are enough for endowing CF1 beta with a reconstitutive capacity, but for exhibition of a significant soluble ATPase activity equivalent amounts of CF1 alpha and beta are required. The CF 1 (alpha beta) complex isolated and purified in this report thus represents the minimal catalytic core of the CF1-ATPase.

Binding of IL-1 beta to alpha-macroglobulins and release by thioredoxin.

Human alpha 2-macroglobulin (H alpha 2M) is a major IL-1 beta binding plasma protein. The characteristics of the H alpha 2M IL-1 beta complex formation suggested, that cleavage of the internal thiol ester in other members of the alpha-macroglobulin family (alpha M) could enable these proteins to bind IL-1 beta. Characterization of optimal conditions for binding 125I IL-1 beta to H alpha 2M showed that H alpha 2M-IL-1 beta complex formation could be obtained over a pH range of 6.3 to 9 in the presence of some metal cations (i.e., Zn2+, Cd2+, Cu2+, Ni2+). Other divalent metal cations (i.e., Mn2+, Mg2+, Ca2+) were without effect. Time kinetic studies showed that binding of IL-1 beta to H alpha 2M was complete within 200 min and that H alpha 2M-IL-1 beta complexes became increasingly resistant to dissociation by boiling in SDS as a function of incubation time. Human pregnancy zone protein, rat alpha 1-, alpha 2-macroglobulin (R alpha 1M, R alpha 2M), all homologous with H alpha 2M, were tested for their ability to bind IL-1 beta. In each instance, alpha M-IL-1 beta complex formation was observed only after treatment of alpha M with methylamine, a primary amine that causes cleavage of the internal thiol ester in alpha M and the appearance of free thiol groups. Similarly, for each of these proteins, complex formation was increased several fold in the presence of Zn2+. Competition experiments using cytokines or proteins of similar molecular mass as IL-1 beta established that only unlabeled IL-1 beta was effective in inhibiting binding of 125I IL-1 beta to H"F" alpha 2M. Acylation of H"F" alpha 2M by diethylpyrocarbonate blocked the binding of IL-1 beta when analyzed by native PAGE. Deacylation of H"F" alpha 2M with hydroxylamine partially restored the binding capacity of H"F" alpha 2M further supporting the involvement of histidyl residues in the Zn2(+)-dependent binding of IL-1 beta. Reduced thioredoxin, but not its alkylated form, from Escherichia coli readily releases H"F" alpha 2M bound IL-1 beta under conditions that did not lead to reduction of disulfide bonds in H"F" alpha 2M. The action of thioredoxin also augmented IL-1-like activity in two independent bioassays suggesting that H"F" alpha 2M bound IL-1 beta is partially biologically inactive or latent. These results suggest that "activated" alpha M exert a modulating role for IL-1 beta by exposing specific binding sites, which are inaccessible in the native proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Active site of bovine adrenocortical cytochrome P-45011.beta. studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450/scc

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)

The 55-kDa polypeptide released from spinach thylakoid membranes with 1 M LiCl is not the beta subunit of chloroplast F1.

It was reported by Frasch et al. (Frasch, W. D., Green, J., Caguiat, J., and Mejia, A. (1989) J. Biol. Chem. 264, 5064-5069) that washing spinach thylakoid membranes with 1 M LiCl caused the release of the beta subunit of chloroplast F1 (CF1) which, existing as 180-kDa complexes of beta 3, retained considerable ATPase activity. We repeated their procedures and confirmed that a CF1 beta-like 55-kDa polypeptide was a major constituent of the 1 M LiCl-washed extract. However, the extract contained another polypeptide of which the Mr was 14,000, and these two polypeptides comprised a complex with approximate Mr 550,000 that had the same mobility in native polyacrylamide gel electrophoresis as that of ribulose-1,5-bisphosphate carboxylase. Only very low ATPase activity, less than 1% of the reported value, was detected for the extract and the purified complex. Antibody against the beta subunit of F1 from a thermophilic bacterium PS3 showed a clear cross-reactivity with the CF1 beta subunit but not with the 55-kDa polypeptide. Analysis of the N-terminal amino acid sequences of the 55- and 14-kDa polypeptides and the whole complex revealed that the complex was ribulose-1,5-bisphosphate carboxylase and that the 55- and 14-kDa polypeptides were its large and small subunits, respectively.

Pre-translational regulation of the (Na+ + K+)-ATPase in response to demand for ion transport in cultured chicken skeletal muscle.

The expression of the (Na+ + K+)-ATPase in cultured chicken skeletal muscle can be altered by varying the demand for ion transport. Veratridine, an activator of voltage-sensitive Na+ channels, causes a specific transient increase in biosynthesis of the sodium pump that accounts for the doubling of the number of (Na+ + K+)-ATPase molecules in the sarcolemma (Wolitzky, B.A., and Fambrough, D.M. (1986) J. Biol. Chem. 261, 9990-9999). Here we report a study of veratridine-induced up-regulation, focusing upon alpha- and beta-mRNA levels and transcription rates. Myotubes normally express the alpha 1-isoform mRNA and the beta-mRNA at a molar ratio of 0.6 +/- 0.1 (S.D.). In the presence of veratridine, the beta-mRNA is transiently up-regulated approximately 3-fold. The kinetics of this increase parallel the rate of beta-subunit protein synthesis. The increase in beta-mRNA during up-regulation is accomplished by an increase in the transcription rate of the beta gene. The veratridine-induced increase in beta-mRNA is not blocked by cycloheximide. The alpha-mRNA also increases during exposure to veratridine, but this increase is very modest and occurs very late in the up-regulation process. The increased beta-mRNA results in over-production of beta-subunits, which we postulate drives more efficient assembly of alpha beta complexes, i.e. sodium pump molecules. As the up-regulated state is achieved the level of beta-mRNA falls abruptly, reflecting a marked decrease in beta-mRNA stability. Treatment of up-regulated myotubes with tetrodotoxin, a veratridine antagonist, results in rapid down-regulation of the sodium pump, while having little or no effect on the levels of alpha 1- and beta-mRNAs.