Kinds Of Subunits Research Articles

Nucleoside diphosphate kinase from human erythrocytes: Purification, molecular mass and subunit structure

A new procedure for the purification of nucleoside diphosphate kinase from human erythrocytes is described. The enzyme (105 kDa by gel filtration) is made up of two different kinds of subunits (19.0 and 20.5 kDa), both displaying enzymatic activity. The probable subunit structure of the enzyme is hexameric. The discrepancies related to earlier work are discussed.

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.

Phosphofructokinase from porcine heart, liver and erythrocytes

1. Phosphofructokinase from porcine heart, liver and erythrocytes were purified by affinity chromatographies on Cibacron Blue Sepharose and N6-ATP agarose. 2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the heart and liver enzymes consist of only one kind of subunit, namely, M and L type subunits, respectively, whereas the erythrocyte enzyme comprises of three kinds of subunits, M, L and C types. 3. Some kinetic and regulatory properties of the enzymes were also measured.

The nitrite oxidizing system ofNitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a1c1 is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c-550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a1c1 and aa3-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c-550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP+ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi. The electron transfer against redox potential from NO2− to cythochrome c could be pushed through prompt removal by cytochrome aa3 of H+ formed by the dehydrogenation of NO2−+ H2O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO2−, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a1c1 with NO2− in vivo.

The nitrite oxidizing system of Nitrobacter winogradskyi

Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a 1 c 1 is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c-550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a 1 c 1 and aa 3-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c-550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP + with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi. The electron transfer against redox potential from NO 2 − to cythochrome c could be pushed through prompt removal by cytochrome aa 3 of H + formed by the dehydrogenation of NO 2 − + H 2O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO 2 −, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a 1 c 1 with NO 2 − in vivo.

Characterization of cyanogenic β-glucosidase (Linamarase) from cassava ( Manihot esculenta Crantz)

Linamarase (EC 3.2.1.21) was purified from cassava petiole, stem, and root cortex by ammonium sulfate precipitation, column chromatography on Sepharose 6B, and chromatofocusing. The last step resolved the enzyme from each source into three forms with p I values of 4.3, 3.3, and 2.9. Each form was found to be oligomeric, consisting of one kind of subunit, M r 63,000. The major isozyme with a p I of 4.3 from petiole showed a K m for linamarin of 0.6 m m and possessed both β-glucosidase and β-fucosidase activities. The former was sensitive to inhibition by δ-gluconolactone, isopropyl-β- d-thioglucoside, and HgCl 2, whereas the latter was inhibited by Tris ion.

Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies

Neuronal acetylcholine receptors (AChRs), which bind nicotine with high affinity but do not bind alpha-bungarotoxin (alpha Bgt), have recently been immunoaffinity-purified from chicken (Whiting and Lindstrom, 1986a) and rat (Whiting and Lindstrom, 1987a) brain using monoclonal antibodies (mAbs). Here we report the characterization of nicotinic AChRs of bovine and human brain using as probes mAbs prepared to AChRs from rat brain. Both the human and bovine brain AChRs exhibit high affinity for L-nicotine (Ki = 16 nM for bovine AChR and Ki = 6.5 nM for human AChR) and other cholinergic agonists, relatively lower affinity for cholinergic antagonists, and do not bind alpha Bgt. These AChRs are affinity-labeled with bromoacetylcholine and 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA) after reduction with dithiothreitol, indicating that amino acid residues homologous to cysteines 192 and 193 of alpha subunits of Torpedo electric organ AChRs are conserved. Immunoaffinity-purified bovine brain AChR consists of 2 types of subunit, Mr 50,600 and Mr 74,400. The Mr 74,400 subunit was affinity-labeled with 3H-MBTA, indicating that it contains the ACh binding site. Thus, mAbs have proven to be excellent probes for these proteins, and have been used to show that neuronal nicotinic AChRs of chickens, rats, and cattle are macromolecules approximately 10 S in size and composed of only 2 kinds of subunits: an ACh-binding subunit and a structural subunit.

CDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA α4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor Cloning of the α-subunit cDNA

Nicotinic acetylcholine receptors (AChRs) from muscle bind α-bungarotoxin (αBgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind αBgt and are composed of two kinds of subunits. αBgt-binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds αBgt. Surprisingly, the AChR of this neuron-derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds αBgt, but is different from αBgt-binding proteins in brain. Here we show that TE671 expresses the α-subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle-type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle-type AChR α-subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis.

Active site organization of bacterial type I fatty acid synthetase.

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.

Characterization of an alkaline proteinase of fish muscle.

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.

Subunit structure and immunological properties of a basic 7s globulin from soybean seeds

Basic 7S globulin from defatted soybean (var. Miyagishirome) meal was isolated from the 0.5 M NaCl-soluble fraction by ion-exchange chromatography on CM-Sepharose CL-6B. The basic 7S globulin was distinguished immunologically from the other soybean proteins. SDS—urea gel electrophoresis of basic 7S globulin gave two kinds of subunits ( M, 16 000 and 26 000), designated as LMWS (low M, subunit) and HMWS (high M, subunit), respectively, in the presence of a 2-mercaptoethanol (2-ME)-containing system. They gave only one intermediary subunit ( M, 42 000) without 2-ME. Two kinds of HMWS (H-I and H-II) and two kinds of LMWS (L-I and L-II) were separated by polyacrylamide gel electrophoresis in a urea-acetic acid system. The basic 7S globulin was fractionated into two components (designated as basic 7S-I and basic 7S-II) on CM-Sepharose CL-6B. The subunits of basic 7S-I and basic 7S-II comprising H-I and L-I, and H-II and L-II, respectively.

Characterization of the amine oxidase involved in the growth of Trichosporon cutaneum X4 on ethylamine as source of carbon, nitrogen and energy.

The amine oxidase from Trichosporon cutaneum X4 grown on ethylamine as carbon, nitrogen and energy source was purified to near homogeneity. The purified enzyme showed the highest resistance to heat of any amine oxidase hitherto characterized from a yeast (half-life at 62 degrees C, 14 min). Measurement of kinetic parameters as a function of carbon chain length showed results typical of a benzylamine oxidase. Both non-denaturing- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed multiple bands, and dimethyl suberimidate cross-linking studies revealed that the enzyme consisted of multimers of two polypeptide chains of Mr respectively 19,000 and 26,000. The smallest structure to show activity probably contained two of each kind of subunit.

Nitrile hydratase of Brevibacterium R312 — purification and characterization —

Nitrile hydratase was purified and crystallized from the crude extract of Brevibacterium R312 and found to be homogeneous by the results of disc gel electrophoresis, analytical ultracentrifuge and double diffusion in agarose. The enzyme has a molecular mass of about 85,000 Da and contains approximately 3 g atoms iron/mol enzyme. The enzyme was composed of two kinds of subunits, of which molecular masses were 26,000 Da and 27,500 Da. The concentrated solution of the enzyme had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 712 nm. The enzyme was active toward various aliphatic nitriles.

Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast.

Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.

57] Proton-conducting portion (F0) from Escherichia coli ATP synthase: Preparation, dissociation into subunits, and reconstitution of an active complex

Publisher Summary This chapter discusses the preparation, dissociation into subunits, and reconstitution of an active complex for the proton-conducting portion (F 0 ) from Escherichia coli ATP synthase. The F 0 part of the ATP synthase complex (F 1 F 0 ) of E. coli is composed of three different polypeptides and the complex serves as a proton channel. After reconstitution into liposomes a functional F 0 complex can be tested either by measuring passive proton translocation or by adding back F 1 . Passive proton translocation through F 0 is measured using K+-loaded liposomes. ATPase activity of reconstituted F 1 F 0 complexes is measured by determining the liberated orthophosphate. The chapter outlines the procedure for preparation of F 0 by hydrophobic interaction chromatography. Reconstitution of a functional F 0 complex is only achieved by incorporation of all three kinds of subunits into phospholipid vesicles.

Isolation and Characterization of Globulin from Seeds ofAmaranthus hypochondriacusL.

Seeds of grain amaranths contain a high amount (about 60% of total nitrogen) of albumin and globulin and a trace amount of prolamin. From salt-soluble extracts of A. hypochondriacus seeds, a globulin (440,000 in apparent molecular weight and ) was purified by Sepharose 6B gel and DEAE-cellulose column chromatographies. The protein comprised at least four kinds of subunits whose molecular weights were 36,000, 32,000, 20,000 and 18,000, respectively. The amino acid composition of the globulin was almost similar to those of soybean and oat globulins.

A comparative survey of several bacterial aa3-type cytochrome c oxidases

The aa3-type cytochrome c oxidases purified from Nitrobacter agilis, Thiobacillus novellus, Nitrosomonas europaea, and Pseudomonas AM 1 were compared. They have haem a and copper atom as the prosthertic groups and show α and γ absorption peaks at around 600 and 440 nm, respectively. Each oxidase molecule is composed of two kinds of subunits. The N. agilis oxidase has 2 moles of haem a and 2 atoms of copper in the minimal structural unit composed of one molecule each of the two kinds of subunits, while the T. novellus enzyme seems to contain one molecule of the haem and one atom of the metal in the unit. The N. europaea oxidase shows very low affinity for carbon monoxide. Each oxidase reacts rapidly with some eukaryotic cytochromes c as well as with its native cytochrome c. The cytochrome c oxidase activity of the N. agilis oxidase is 50% inhibited by 1 μM KCN, while 50% inhibition of the activity requires 100 μ KCN in the case of the N. europaea enzyme.

All three subunits are required for the reconstitution of an active proton channel (F0) of Escherichia coli ATP synthase (F1F0).

The membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli is built up from three kinds of subunits a, b and c with the proposed stoichiometry of 1:2:10 +/- 1. We have dissociated the F0 complex by treatment with trichloroacetate (3 M) at pH 8.0, in the presence of deoxycholate (1%) and N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-14, 5%). The subunits were separated by gel filtration with trichloroacetate (1 M) included in the elution buffer. The homogeneity of the fractions was checked by rechromatography and SDS-gel electrophoresis. After integration into phospholipid vesicles each subunit alone as well as all possible combinations were tested for H+ translocating activity and binding of F1. A functional H+ channel could only be reconstituted by the combination a1b2c10 which corresponds to that of native F0.

Isolation and characterization of globulin from seeds of Amaranthus hypochondriacus L.

Seeds of grain amaranths contain a high amount (about 60% of total nitrogen) of albumin and globulin and a trace amount of prolamin. From salt-soluble extracts of A. hypochondriacus seeds, a globulin (440, 000 in apparent molecular weight and s020, w= 12-7) was purified by Sepharose 6B gel and DEAE-cellulose column chromatographies. The protein comprised at least four kinds of subunits whose molecular weights were 36, 000, 32, 000, 20, 000 and 18, 000, respectively. The amino acid composition of the globulin was almost similar to those of soybean and oat globulins.