Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Cooperativity in the dopamine beta-monooxygenase reaction. Evidence for ascorbate regulation of enzyme activity

The steady-state kinetic behavior of dopamine beta-monooxygenase (D beta M) has been examined over a 1000-fold range of ascorbate concentrations. Kinetic plots exhibit extreme curvature indicative of apparent negative cooperativity in the interaction of D beta M with ascorbate, with a calculated Hill coefficient of 0.15-0.30. The observed cooperativity is found to be independent of enzyme concentration and tyramine and oxygen concentrations, as well as the pH employed for the assay. Similar kinetic data have been obtained with both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion activator fumarate upon the observed kinetic patterns has demonstrated a conversion to a less cooperative kinetic pattern at low pH and high concentrations of fumarate. This phenomenon is attributed to an inhibitory binding of the structurally similar monoanionic species of fumarate to the ascorbate reductant site. A simple model has been used to assess the change in apparent Vmax and Km parameters with increased ascorbate concentrations. At all pH values examined, there is a dramatic decrease in the affinity of D beta M for ascorbate from a Km of approximately 0.05-0.10 mM (ascorbate concentration less than 1 mM) to Km greater than 10 mM at limiting ascorbate; at the same time there is a 3- to 4-fold increase in the limiting Vmax value. Several models have been considered to explain the observed activation of D beta M by high levels of ascorbic acid.     

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex.

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).     

Isolation and sequencing of cDNAs for the XL-I and XL-III forms of bovine liver xenobiotic-metabolizing medium-chain fatty acid:CoA ligase

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was previously purified to apparent homogeneity from bovine liver mitochondria, and the amino acid sequence of a short segment of the enzyme was determined. This sequence was used to develop a probe for screening a bovine cDNA library from which a 1.6 kb cDNA was isolated. This cDNA was sequenced and found to contain the code for the known amino acid sequence. The complete open reading frame was not present in this cDNA, but it was estimated to code for approximately 75% of the XL-I sequence. The XL-III ligase was purified to apparent homogeneity from bovine liver mitochondria. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. The enzyme was chemically cleaved using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides of ca. 21 and 25 kDa, plus several smaller peptides including a prominent 6 kDa peptide. The N-terminus of the 6, 21, and 25 kDa peptides was sequenced and the 21 and 25 kDa sequences were identical indicating incomplete cleavage. The sequences were used to design probes for screening a bovine liver cDNA library. This resulted in the isolation of a 2,065 bp cDNA. This cDNA was sequenced and found to contain the initiation and termination codons, as well as the requisite amino acid sequences. The open reading frame coded for a 64,922 Da protein. The sequence of XL-III cDNA was markedly different from that of XL-I, indicating the genetic uniqueness of the two ligases. They are, however, 64% homologous, which suggests a common evolutionary origin.     

Soluble bovine galactose-binding lectin. cDNA cloning reveals the complete amino acid sequence and an antigenic relationship with the major encephalitogenic domain of myelin basic protein.

A full-length cDNA clone for the 13-14 kDa soluble beta-galactoside-binding lectin was isolated from a bovine fibroblast cDNA library. The derived amino acid sequence shows eight differences from a preliminary partial amino acid sequence given previously for the bovine heart lectin. This observation led to a re-examination of the data and correction of the heart lectin protein sequence. Except for a possible polymorphism of the heart lectin at position 57, the fibroblast and heart lectin sequences are considered identical. The epitope recognized by two monoclonal anti-(bovine lectin) antibodies, 36/8 and 9/5, was identified as the tetrapeptide sequence W-G-A/S-E/D by the isolation of several different cDNA clones from a human intestine cDNA library. A similar tetrapeptide is present in all of the soluble beta-galactoside-binding animal lectins sequenced thus far. It is also found in myelin basic protein, which we show is antigenically cross-reactive with the lectin. In myelin basic protein the tetrapeptide is a part of the major domain previously shown to be responsible for the induction of experimental allergic encephalomyelitis.     

Purification of bovine cholecystokinin-58 and sequencing of its N-terminus

Molecular cloning of cholecystokinin (CCK) mRNA from porcine brain and gut has demonstrated that CCK is synthesized as an identical precursor in both tissues. The sequence for porcine CCK-58 predicted from CCK cDNA was identical with the amino acid sequence of the peptide purified from different lots of animals. However one group did report that there were differences in the N-terminus of CCK-58 purified from the intestines of two different lots of mongrel dogs. In the current report it is demonstrated that the amino acid sequences of CCK-58 purified separately from three bovine brains are identical through the first 19 N-terminal amino acid residues. The peptides were sequenced for ten additional steps and were shown to be identical with the previously reported sequences for the N-terminus of CCK-39. The N-terminus of bovine CCK-58 has the following sequence: AVPRVDDEPRAQLGALLAR.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.     

Cloning and sequencing of a full-length cDNA of mouse N-acetylglucosamine (beta 1-4)galactosyltransferase

A full-length cDNA clone for mouse N-acetylglucosamine (beta 1-4)galactosyltransferase (beta 1-4GT) [EC 2.4.1.90] and several clones diverged from the beta 1-4GT cDNA were isolated from a mouse F9 cDNA library and then sequenced. The beta 1-4GT cDNA has an open reading frame consisting of 399 amino acids. The homology at the amino acid level is 80 and 91% as to the partial sequences of bovine and human milk beta 1-4GT, respectively. The general enzyme structure of the beta 1-4GT seems to be similar to that of a rat beta-galactoside (alpha 2-6) sialyltransferase. Junctions of the common and divergent regions of cDNA have dinucleotides, AG, suggesting that the variety of cDNA clones is generated through alternative splicing.     

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

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

Isolation and characterization of cDNAs encoding beta A2- and beta A4-crystallins: heterologous interactions in the predicted beta A4-beta B2 heterodimer

Except for the two acidic chains, beta A2 and beta A4, the primary structures of all bovine beta-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both beta A2 and beta A4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. beta A2, comprising 197 aa, and beta A4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of beta A4 was made based on the coordinates of one subunit from the beta B2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine beta-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic beta-crystallins with a view to defining structural features necessary for subunit recognition in beta-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine beta-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic beta-crystallins.     

cDNA clone to chick corneal chondroitin/dermatan sulfate proteoglycan reveals identity to decorin.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.     

Isolation,sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria

The purification of xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM-A and HXM-B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N-terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM-A and HXM-B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full-length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64230-Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium-chain acyl-CoA synthetase) enzyme, 58.7% homology to the bovine XL-III XM-ligase, and 81.5% homology to the bovine XL-I XM-ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM-A.     

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's A beta(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [A beta1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of A beta(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, A beta(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. A beta(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, A beta(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the A beta-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, A beta(1-42)D1Nle, completely abrogated the oxidative stress and neurotoxicity associated with the A beta(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.     

Amino acid and cDNA sequence of bovine phosducin, a soluble phosphoprotein from photoreceptor cells

Vertebrate photoreceptor cells contain a soluble phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the GTP-binding protein, transducin. Light-induced changes in cyclic nucleotide levels modulate the phosphorylation of phosducin by protein kinase A. The complete amino acid sequence of purified phosducin from bovine retinas was determined by Edman degradation from overlapping polypeptides derived from enzymatic digestion by trypsin and Staphylococcus aureus V8 protease or from chemical degradation by cyanogen bromide. Excluding the unidentified group which blocks the NH2 terminus, phosducin contains 245 amino acids with a calculated molecular weight of 28,185 and isoelectric point of pH 4.5. Phosducin is enriched with acidic and sulfur-containing amino acids, having 32 glutamic acid, 16 aspartic acid, 9 methionine, and 5 cysteine residues. It also contains 24 serine and 8 threonine residues, of which only serine 73 is located within a consensus phosphorylation sequence (-RKMS(P)QV-) for cyclic nucleotide-dependent protein kinase. Secondary structure analysis predicts the presence of 62% alpha-helix, 22% beta-sheet, and 16% random coil, with eight turns. Computer-aided searches of protein data banks revealed no apparent homology to any sequenced protein except that coded by a MEKA cDNA clone (Kuo, C-H., Akiyama, M., and Miki, N. (1989) Mol. Brain Res. 6, 1-10) which deviates from the confirmed phosducin sequence in the last 15 amino acids. Sequence analysis of a cDNA clone for bovine retinal phosducin confirmed that the MEKA clone deviation resulted from an unidentified cDNA guanosine nucleotide, a shifted reading frame and a premature stop codon.     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

Nucleotide sequence of ovine macrophage interleukin-1 beta cDNA.

We have cloned and sequenced the cDNA for the coding region of ovine alveolar macrophage interleukin-1 beta. At the nucleotide level, the ovine cDNA shares 95, 74 and 71% homology with the bovine, human and murine cDNA equivalents or homologs. Comparison at the amino acid level revealed 95% homology with bovine IL-1 beta and approximately 57% with the human and murine homologs.     

Frog lens beta A1-crystallin: the nucleotide sequence of the cloned cDNA and computer graphics modelling of the three-dimensional structure

Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.     

Binding of pyridine coenzymes to the beta-subunit of the voltage sensitive potassium channels

The beta-subunit of the voltage-sensitive K(+) channels shares 15-30% amino acid identity with the sequences of aldo-keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K(v)beta2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K(D) values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1+/-0.007 and 0.05+/-0.006 M, respectively - indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the alpha-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K(D) for NADH and NAD were between 1-3 microM whereas the K(D) for FAD was >10 microM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2' phosphate at the binding site. The selectivity of K(v)beta2.1 for NADPH over NADP may be significant in regulating the K(+) channels as a function of the cellular redox state.     

Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine.

A cDNA clone encoding the N-terminal sequence of the murine integrin beta 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa.