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.     

Structure of the protein phosphatase 2A holoenzyme

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.     

Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme.

Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce the loss of kinase activity, strongly suggesting a protective function for the beta subunit. Assaying different peptides for specificity toward the recombinant alpha subunit and the recombinant reconstituted enzyme, showed that the presence of the beta subunit could modify the specificity of the catalytic alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit, counteracts the stimulatory effect of the beta subunit on the alpha subunit activity and partially substitutes the beta subunit in conferring thermal stability to the alpha subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the beta subunit including the autophosphorylation site. It is suggested that the acidic domain of the beta subunit, encompassing residues 55-71, plays a role in the interactions between the beta and alpha subunits.     

Detection and purification of the free A subunit of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli

After removal of total B subunit and heat-labile enterotoxin (LT) from crude cell extracts of enterotoxigenic Escherichia coli (HB 101-EWD 299) by Bio-gel A 5 m column chromatography, the crude cell extract was shown to contain a free A subunit (A' subunit) that did not bind to the coligenoid of the B subunits. The A' subunit was found to be immunologically identical to the A subunit of holo-LT and was purified to show only one band in SDS-poly-acrylamide gel electrophoresis (PAGE). The mobility of the A' subunit was identical to that of the A subunit of holo-LT. The pI value of the A' subunit was also the same as that of the A subunit of holo-LT. These data suggest that in enterotoxigenic E. coli there is free A subunit which may be involved in formation of holo-LT, analogously to free B subunit (coligenoid), and that the free A subunit is physicochemically and immunologically identical to the A subunit of holo-LT.     

S100a0 (αα) Protein Is Present in Neurons of the Central and Peripheral Nervous System

The cellular distribution of S100 subunits in human brain and peripheral nerves was studied by means of an immunohistochemical technique using antibodies specific to the alpha subunit or the beta subunit of S100 protein. The results indicate that the distribution of the alpha subunit and the beta subunit is different among cell types in the nervous tissue, and that neurons in the brain and peripheral nerves contain only the alpha subunit, or S100a0 protein. The subunit distribution also appears to be different at an intracellular level, where the immunoreaction products for the alpha subunit show granular arrangement whereas those for the beta subunit are found diffusely in the cytoplasm.     

Identification of the structural subunits required for formation of the metal centers in subunit I of cytochrome c oxidase of Rhodobacter sphaeroides

Genetic manipulation of the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I(a)()) that contained a single heme A. No copper was present in this protein, indicating that the heme a(3)-Cu(B) active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I-II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I(a)(). Since significant amounts of subunit I(a)() were also isolated from wild-type cells, it is likely that subunit I(a)() will be present in any preparation of the aa(3)-type oxidase isolated via an affinity tag on subunit I.     

Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.     

小麦高分子量谷蛋白亚基效应的比较研究

采用SDS-PAGE方法,通过对5个亲本间杂交获得的F2群体每一单株的F3籽粒样本及其亲本进行小麦高分子量谷蛋白亚基（HMW-GS）组成分析,并对每一F2单株上F3籽粒群体的高分子量谷蛋白亚基组成与其籽粒蛋白质含量、SDS-沉降值的关系进行研究,分析比较黄淮麦区出现频率较高的7个亚基或亚基对的品质效应。结果表明：小麦高分子量谷蛋白亚基组成不同群体间籽粒的蛋白质含量和SDS-沉降值基本达到显著或极显著水平。优质亚基表现为：1、7＋8、14＋15和5＋10亚基。因此,黄淮麦区小麦育种应加强对这些优质亚基的引入和利用,特别是对14＋15和5＋10亚基的引入和利用。     

Identification, characterization, and functional analysis of heart-specific myosin light chain phosphatase small subunit

Myosin light chain phosphatase consists of three subunits, a 38-kDa catalytic subunit, a large 110-130-kDa myosin binding subunit, and a small subunit of 20-21 kDa. The catalytic subunit and the large subunit have been well characterized. The small subunit has been cloned and studied from smooth muscle, but little is known about its function and specificity in the other muscles such as cardiac muscle. In this study, cDNAs for heart-specific small subunit isoforms, hHS-M(21), were isolated and characterized. Evidence was obtained from an analysis of genome to suggest that the small subunit was the product of the same gene as the large subunit. Using permeabilized renal artery preparation and permeabilized cardiac myocytes, it was shown that the small subunit increased sensitivity to Ca(2+) in muscle contraction. It was also shown using an overlay assay that hHS-M(21) bound the large subunit. Mapping experiments demonstrated that the binding domain and the domain involved in the increasing Ca(2+) sensitivity mapped to the same N-terminal region of hHS-M(21). These observations suggest that the heart-specific small subunit hHS-M(21) plays a regulatory role in cardiac muscle contraction by its binding to the large subunit.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.     

Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.     

A new protein subunit k for RNA polymerase from Xanthomonas campestris pv. oryzae

During the purification of RNA polymerase from Xanthomonas campestris pv. oryzae, a new subunit named k was found to be associated with this enzyme. The removal of subunit k from holoenzyme by DEAE-cellulose column chromatography results in a decrease in specific activity of the enzyme. The readdition of subunit k to subunit k-depleted holoenzyme results in restoration of enzymatic activity. Subunit k increase the activity of RNA polymerase; the activation was in proportion to the concentration of subunit k added. Antiserum against holoenzyme devoid of subunit k was prepared. This antiserum did not react with purified subunit k; therefore, subunit k may not be the proteolytic fragment of the beta, beta', sigma, or alpha subunit. When this antiserum was used to precipitate RNA polymerase obtained from a crude extract of bacterial cells, subunit k was coprecipitated as determined by sodium dodecyl sulfate gel electrophoretic analysis. The molecular mass of subunit k is approximately 29 kDa, and the molar ratio of beta:beta':sigma:alpha:k was estimated to be 1:1:1:2:4. When native Xp10 DNA was used as template, subunit k stimulated subunit k-depleted holoenzyme, but not core enzyme. When the synthetic polynucleotide poly[d(A-T)] was used, subunit k activated both subunit k-depleted holoenzyme and core enzyme. Subunit k also activated the binding of RNA polymerase to template DNA.     

Estimation of the gene frequency of lactate dehydrogenase subunit deficiencies

To detect the frequency of lactate dehydrogenase (LDH) subunit deficiency, screening for LDH subunit deficiency was performed on 3,776 blood samples from healthy individuals in Shizuoka Prefecture by means of electrophoresis. The frequency of heterozygote with LDH-A subunit deficiency was found to be 0.185%, and with LDH-B subunit deficiency, 0.159%. The frequencies of both subunit deficiencies were not significantly different. Gene frequencies of LDH subunit deficiencies were calculated by the simple counting procedure, and the results are as follows: gene frequency of LDH-A subunit deficiency was 11.9 X 10(-4), and that of LDH-B subunit deficiency, 7.9 X 10(-4). In addition, the second case in the world of a homozygous individual with LDH-A subunit deficiency was detected by this screening. This case with regard to the characteristics of LDH-A subunit deficiency are summarized herein.     

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Altered Localization of the δ Subunit of the GABAA Receptor in the Thalamus of α4 Subunit Knockout Mice

The α4 subunit of the GABA_A receptor (GABA_AR) is highly expressed in the thalamus where receptors containing the α4 and δ subunits are major mediators of tonic inhibition. The α4 subunit also exhibits considerable plasticity in a number of physiological and pathological conditions, raising questions about the expression of remaining GABA_AR subunits when the α4 subunit is absent. Immunohistochemical studies of an α4 subunit knockout (KO) mouse revealed a substantial decrease in δ subunit expression in the ventrobasal nucleus of the thalamus as well as other forebrain regions where the α4 subunit is normally expressed. In contrast, several subunits associated primarily with phasic inhibition, including the α1 and γ2 subunits, were moderately increased. Intracellular localization of the δ subunit was also altered. While δ subunit labeling was decreased within the neuropil, some labeling remained in the cell bodies of many neurons in the ventrobasal nucleus. Confocal microscopy demonstrated co-localization of this labeling with an endoplasmic reticulum marker, and electron microscopy demonstrated increased immunogold labeling near the endoplasmic reticulum in the α4 KO mouse. These results emphasize the strong partnership of the δ and α4 subunit in the thalamus and suggest that the α4 subunit of the GABA_AR plays a critical role in trafficking of the δ subunit to the neuronal surface. The findings also suggest that previously observed reductions in tonic inhibition in the α4 subunit KO mouse are likely to be related to alterations in δ subunit expression, in addition to loss of the α4 subunit.     

Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.