Partial amino acid sequence of a hemocyanin subunit from Palinurus vulgaris

1. Hemocyanin from the spiny lobster Palinurus vulgaris was separated into two fractions, which were designated as subunits a and b. 2. 55% of the amino acid sequence of subunit b has been determined. A comparison with Panulirus interruptus hemocyanins shows 78% sequence identity with subunit a and 56% with subunit c. It has carbohydrate attached to domain one. Two half-cystines have been substituted, indicating that it probably possesses only one disulfide bridge. Heterogeneity has been observed in seven out of 380 positions determined so far. 3. Subunit a is almost identical with subunit b. In contrast to Panulirus interruptus and Panulirus japonicus, Palinurus vulgaris hemocyanin contains no c-type subunit. 4. A position in the tentative evolutionary tree of arthropod hemocyanins based on sequence differences has been assigned to Palinurus vulgaris subunit b.     

ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.     

Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.     

Both the p33 and p55 Subunits of the Helicobacter pylori VacA Toxin Are Targeted to Mammalian Mitochondria

Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.     

Three-dimensional structure of pig muscle phosphoglucose isomerase at 6 A resolution

A 6 Å resolution electron density map of pig muscle phosphoglucose isomerase has been obtained. From this map it has been possible to isolate a single molecule and to assign tentative subunit boundaries. The binding of the competitive inhibitor 6-phosphogluconate has been studied. The binding site appears to be close to the subunit interface.     

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.     

Further studies on partially purified calf thymus DNA polymerase a.

Attempts to prevent the urea conversion of a 200-230,000 molecular weight DNA polymerase alpha to a 150-170,000 molecular weight form by the inclusion of protease inhibitors have not been successful. No other method has been found capable of dissociating a 50-70,000 fragment or subunit from the DNA polymerase subunit. Addition of this 50-70,000 subunit to the polymerase subunit does not aid the binding of the enzyme to DNA, but does have an effect on the utilisation of synthetic template-initiator complexes by the polymerase subunit.     

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.     

Mutants of Escherichia coli H+-ATPase defective in the delta subunit of F1 and the b subunit of F0

Complete nucleotide sequence of the genes for subunits of the H+ ATPase of E.coli has been determined and several hybrid plasmids carrying various portions of these genes have been constructed. Genetic complementation and recombination tests of about forty mutants of E.coli defective in the ATPase were performed using these plasmids for identifying the locations of the mutations. Two mutants defective in the delta subunit and a novel type of mutant defective in the b subunit of F0 were identified. The delta subunit mutants showed no proton conduction, suggesting that this subunit has an important role for the proton conduction. The ATPase of the b subunit mutant has a normal activity of proton channel portion, which phenotype is clearly different from that of mutants of the b subunit reported previously.     

Cloning and characterization of the yeast nuclear gene for subunit 5 of cytochrome oxidase

The nuclear gene COX5 coding for subunit 5 of cytochrome oxidase has been cloned by transformation of the cox5-1 mutant aE4-238/AL1 with a library of yeast genomic DNA. The recombinant plasmid pG46/ST2 bearing a nuclear DNA insert of 1.17 kilobase pairs restores the ability of cox5 mutants to respire and to synthesize a wild type subunit 5. The COX5 gene has been sequenced and determined to code for a 153-amino acid long protein with a molecular weight of 17,121. The amino-terminal 20 residues comprise the signal peptide. The sequence starting from residue 21 matches the partial sequence reported for the mature subunit 5. The sequence of the subunit 5 gene indicates that the mature protein has a molecular weight of 14,858 which agrees with previous size estimates based on electrophoretic migration. The primary sequence and polarity profile of yeast subunit 5 establishes that it is homologous to subunit 4 of bovine cytochrome oxidase.     

Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1

The thermal stability of cholera toxin free in solution and in association with its cell-surface receptor ganglioside GM1 has been studied by using high-sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. In the absence of ganglioside GM1, cholera toxin undergoes two distinct thermally induced transitions centered at 51 and 74 degrees C, respectively. The low-temperature transition has been assigned to the irreversible thermal denaturation of the active A subunit. The second transition has been assigned to the reversible unfolding of the B subunit pentamer. The isolated B subunit pentamer exhibits a single transition also centered at 74 degrees C, suggesting that the attachment of the A subunit does not contribute to the stability of the pentamer. In the intact toxin, the A subunit dissociates from the B subunit pentamer at a temperature that coincides with the onset of the B subunit thermal unfolding. In aqueous solution, the denatured A subunit precipitates after dissociation from the B subunit pentamer. This phenomenon can be detected calorimetrically by the appearance of an exothermic heat effect. In the presence of ganglioside GM1, the B subunit is greatly stabilized as indicated by an increase of 20 degrees C in the transition temperature. In addition, ganglioside GM1 greatly enhances the cooperative interactions between B subunits. In the absence of ganglioside, each monomer within the B pentamer unfolds in an independent fashion whereas the fully ganglioside-bound pentamer behaves as a single cooperative unit. On the contrary, the thermotropic behavior of the A subunit is only slightly affected by the presence of increasing concentrations of ganglioside GM1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.     

Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits

Near-full-length cDNA clones to the small and large subunit of the heterotetrameric potato tuber ADP-glucose pyrophosphorylase have been isolated and characterized. The missing amino terminal sequence of the small subunit has also been elucidated from its corresponding genomic clone. Primary sequence comparisons revealed that each potato subunit had less identity to each other than to their homologous subunit from other plants. It also appeared that the smaller subunit is more conserved among the different plants and the larger subunit more divergent. Amino acid comparisons of both potato tuber sequences to theEscherichia coli ADP-glucose pyrophosphorylase sequence revealed conserved regions important for both catalytic and allosteric function of the bacterial enzyme.     

Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis

Among the laccases produced by the white-rot fungus Pleurotus ostreatus, there are two closely related atypical isoenzymes, POXA3a and POXA3b. These isoenzymes are endowed with quaternary structure, consisting of two subunits very different in size. The POXA3 large subunit is clearly homologous to other known laccases, while the small subunit does not show significant homology with any protein in data banks. To investigate on the singular structure of the POXA3 complex, a new system for recombinant expression of heterodimer proteins in the yeast Kluyveromyces lactis has been set up. A unique expression vector has been used and the cDNAs encoding the two subunits have been cloned under the control of the same bi-directionally acting promoter. Expression of the large subunit alone and co-expression of both subunits in the same host have been demonstrated and the properties of the recombinant proteins have been compared. Clones expressing the large subunit alone exhibited always notably lower activity than those expressing both subunits. In addition to the activity increase, the presence of the small subunit led to a significant increase of laccase stability. Therefore, a role of the small subunit in POXA3 stabilisation is suggested.     

Intersubunit interactions in proton-translocating adenosine triphosphatase as revealed by hydrogen-exchange kinetics

The rates of hydrogen-deuterium exchange in the peptide groups of the alpha and beta subunits and the alpha-beta subunit complex of proton-translocating adenosine triphosphatase from the thermophilic bacterium PS3 were examined. The exchange was found to be much slower in the isolated beta subunit than in the isolated alpha subunit. This has been taken as indicating that the structure of the beta subunit is tighter than that of the alpha subunit. Adenosine 5'-triphosphate (ATP) caused tightening of a relatively tight portion of the alpha subunit and of a relatively loose portion of the beta subunit. When the alpha and beta subunits are brought into contact, tightening of the alpha subunit, but not the beta subunit, occurs. The effect of ATP on the structure of the beta subunit is more pronounced in the presence of the alpha subunit than in its absence. These findings support the idea proposed previously that the alpha subunit has an allosteric site and the beta subunit a catalytic site and that the conformation of the beta subunit is controlled by the alpha subunit.     

Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.     

Sulfhydryl groups of cyclic AMP-dependent histone kinase from pig brain]

A preparative method of isolation of the histone kinase regulatory subunit has been developed. The total number of cysteine residues in the enzyme (13 residues) and in each of its subunits (3 residues in the catalytic subunit and 10 residues in the regulatory one), has been determined. Two disulfide bridges have been found in the regulatory subunits. The relative availability of the sulfhydryl groups with respect to different modifying agents has been studied. It has been demonstrated that SH-groups blocking does not change essentially the enzyme activity.     

A method for assessing the reassembly of a multisubunit glycoprotein by western blotting

The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, alpha and beta. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the alpha subunit or hCG; B105, which binds the beta subunit or hCG; and B107, specific for the intact hCG dimer. The alpha subunit and beta subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the alpha subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant alpha subunit was determined to have additional carbohydrate which interfered with the binding of the beta subunit. N-Glycanase digestion of the recombinant alpha subunit produced a form which, when incubated with the beta subunit, did recombine on the nitrocellulose and could be recognized by B107.     

Conserved Amphipathic Helices Near the N-Terminus and C-Terminus of the Alpha Subunit of Cranin (Dystroglycan)

Cranin (dystroglycan) is a ubiquitously expressed extracellular matrix receptor, synthesized as a single precursor, which is cleaved into an extracellular subunit (alpha) and a transmembrane subunit (beta). The primary sequence of cranin (dystroglycan) is known from cDNA cloning, and the protein has been strongly implicated in morphogenesis, cell adhesion and human disease. Nevertheless, the domain structure of the alpha subunit has not been well studied; although the protein binds to matrix proteins, to the beta subunit. to cell surfaces, and possibly to other membrane proteins such as sarcoglycans, the domains responsible for mediating these interactions remain unknown. Here I report computer analyses that identify two distinctive amphipathic alpha-helical regions near the N-terminus and C-terminus of the alpha subunit, which are conserved in all species for which sequence information is currently available. This finding should stimulate and guide experimental studies designed to understand how the alpha subunit is associated with the cell surface and with its various ligands.