scholarly journals Molecular cloning and deduced amino acid sequences of the α- and β- subunits of mammalian NAD+-isocitrate dehydrogenase

1995 ◽  
Vol 310 (3) ◽  
pp. 917-922 ◽  
Author(s):  
B J Nichols ◽  
A C F Perry ◽  
L Hall ◽  
R M Denton
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Isocitrate Dehydrogenase ◽  
Sequence Data ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Peptide Sequence ◽  
Alpha Subunit ◽  
Cdna Clones ◽  
Oligonucleotide Primers

A 153 bp fragment of the cDNA encoding the beta-subunit of pig heart NAD(+)-isocitrate dehydrogenase (NAD(+)-ICDH) was specifically amplified by PCR, using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate cDNA clones encoding the complete mature form of the beta-subunit from a monkey testis cDNA library. Examination of the deduced amino acid sequence of the monkey subunit and the partial sequence of the pig heart enzyme revealed a high level of sequence conservation. In addition, 3 overlapping fragments of the cDNA for the alpha-subunit of monkey NAD(+)-ICDH were amplified using oligonucleotide primers derived from the cDNA sequence of a subunit of bovine NAD(+)-ICDH (EMBL accession no: U07980). These cDNA fragments allow deduction of the amino acid sequence of the alpha-subunit. Since the gamma-subunit of monkey NAD(+)-ICDH has already been cloned [Nichols, Hall, Perry and Denton (1993) Biochem. J. 295, 347-350], a deduced amino acid sequence is now available for all three subunits of mammalian NAD(+)-ICDH. Interrelationships between these subunits are discussed and they are compared with the two subunits of yeast NAD(+)-ICDH and Escherichia coli NADP(+)-ICDH.

scholarly journals Amino acid sequence of the human fibronectin receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1183-1190 ◽  
Author(s):  
W S Argraves ◽  
S Suzuki ◽  
H Arai ◽  
K Thompson ◽  
M D Pierschbacher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Calcium Binding ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Receptor Subunit ◽  
Fibronectin Receptor ◽  
Adhesion Receptor ◽  
Receptor Beta

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.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases

1995 ◽  
Vol 108 (1) ◽  
pp. 17-24 ◽  
Author(s):  
S.M. Hughes ◽  
K.T. Vaughan ◽  
J.S. Herskovits ◽  
R.B. Vallee
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cytoplasmic Dynein ◽  
Peptide Sequence ◽  
Organelle Transport ◽  
Cdna Clones ◽  
Post Translational Modification ◽  
Sequence Element ◽  
Transporter Family ◽  
Intermediate Chains

Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermediate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53–59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of approximately 55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.

scholarly journals Molecular cloning and deduced amino acid sequences of the γ-subunits of rat and monkey NAD+-isocitrate dehydrogenases

1993 ◽  
Vol 295 (2) ◽  
pp. 347-350 ◽  
Author(s):  
B J Nichols ◽  
L Hall ◽  
A C F Perry ◽  
R M Denton
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Amino Acid Sequences ◽  
Peptide Sequence ◽  
Cdna Libraries ◽  
Gamma Subunit ◽  
Gamma Subunits ◽  
Testis Cdna ◽  
Rat Epididymis ◽  
High Level

A 600 bp cDNA fragment encoding part of the gamma-subunit of pig heart NAD(+)-isocitrate dehydrogenase (ICDH gamma) was amplified by PCR using redundant oligonucleotide primers based on partial peptide sequence data [Huang and Colman (1990) Biochemistry 29, 8266-8273]. This PCR fragment was then used as a probe to isolate clones encoding the complete mature forms of the gamma-subunit from rat epididymis and monkey testis cDNA libraries. Comparison of the deduced amino acid sequences of the rat and monkey subunits and the partial sequence of the pig heart enzyme revealed a remarkably high level of sequence identity. The relationship between the deduced amino acid sequences of the NAD(+)-ICDH gamma-subunits and those of nonmammalian NAD(+)- and NADP(+)-ICDH subunits is discussed.

scholarly journals Purification, characterization and cDNA cloning of human lung surfactant protein D

1992 ◽  
Vol 284 (3) ◽  
pp. 795-802 ◽  
Author(s):  
J Lu ◽  
A C Willis ◽  
K B M Reid
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Human Lung ◽  
Solid Phase ◽  
Surfactant Protein ◽  
Amino Acid Sequences ◽  
Surfactant Protein D ◽  
Cdna Clones ◽  
Oligonucleotide Probes

Human pulmonary surfactant protein D (SP-D) was identified in lung lavage by its similarity to rat SP-D in both its molecular mass and its Ca(2+)-dependent-binding affinity for maltose [Persson, Chang & Crouch (1990) J. Biol. Chem. 265, 5755-5760]. For structural studies, human SP-D was isolated from amniotic fluid by affinity chromatography on maltose-Sepharose followed by f.p.l.c. on Superose 6, which showed it to have a molecular mass of approx. 620 kDa in non-dissociating conditions. On SDS/PAGE the human SP-D behaved as a single band of 150 kDa or 43 kDa in non-reducing or reducing conditions respectively. The presence of a high concentration of glycine (22%), hydroxyproline and hydroxylysine in the amino acid composition of human SP-D indicated that it contained collagen-like structure. Collagenase digestion yielded a 20 kDa collagenase-resistant globular fragment which retained affinity for maltose. Use of maltosyl-BSA as a neoglycoprotein ligand in a solid-phase binding assay showed that human SP-D has a similar carbohydrate-binding specificity to rat SP-D, but a clearly distinct specificity from that of other lectins, such as conglutinin, for a range of simple saccharides. Amino acid sequence analysis established the presence of collagen-like Gly-Xaa-Yaa triplets in human SP-D and also provided sequence data from the globular region of the molecule which was used in the synthesis of oligonucleotide probes. Screening of a human lung cDNA library with the oligonucleotide probes, and also with rabbit anti-(human SP-D), allowed the isolation of two cDNA clones which overlap to give the full coding sequence of human SP-D. The derived amino acid sequence indicates that the mature human SP-D polypeptide chain is 355 residues long, having a short non-collagen-like N-terminal section of 25 residues, followed by a collagen-like region of 177 residues and a C-terminal C-type lectin domain of 153 residues. Comparison of the human SP-D and bovine serum conglutinin amino acid sequences indicated that they showed 66% identity despite their marked differences in carbohydrate specificity.

scholarly journals Binding Interaction of a Ring-hydroxylating Dioxygenase with Fluoranthene in Pseudomonas aeruginosa DN1

2021 ◽  
Author(s):  
Shu-Wen Xue ◽  
Yue-Xin Tian ◽  
Jin-Cheng Pan ◽  
Ya-Ni Liu ◽  
Yanling Ma
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Gene Knockout ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Binding Interaction ◽  
Mononuclear Iron ◽  
Genes Encoding ◽  
Substrate Conversion

Abstract Pseudomonas aeruginosa DN1 can efficiently utilize fluoranthene as its sole carbon source, and the first step in the biodegradation process is catalyzed by a ring-hydroxylating dioxygenase (RHD). To better understand the binding interaction of RHD with fluoranthene in the strain DN1, the genes encoding alpha subunit (RS30940) and beta subunit (RS05115) of the RHD were functionally characterized using gene knockout approach and homology modeling combined with molecular docking. The results showed that the mutants lacking the characteristic alpha subunit and/or beta subunit failed to degrade fluoranthene effectively. Based on the translated protein sequence and Ramachandran plot, 96.5 % of the primary amino-acid sequences of the alpha subunit in the modeled structure of the RHD were in the permitted region, 2.3 % in the allowed region, but 1.2 % in the disallowed area. The active center of the alpha subunit constituted a triangle structure of the mononuclear iron atom and the two oxygen atoms coupled with a catalytic ternary of His217-His222-Asp372 for the dihydroxylation reaction with fluoranthene. Amino acid residues adjacent to fluoranthene were nonpolar groups, and the C7-C8 positions on the fluoranthene ring were estimated to be the best oxidation sites. The distance of C7-O and C8-O was 3.77 Å and 3.04Å respectively, and both of them were parallel. The results demonstrated that the dihydroxylation reaction was initiated at C7-C8 positions of the fluoranthene ring by RHD in P. aeruginosa DN1, indicating that the binding interaction may be useful for predicting substrate conversion of RHDs.

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs

1993 ◽  
Vol 13 (3) ◽  
pp. 1385-1391
Author(s):  
H Watanabe ◽  
J Sawada ◽  
K Yano ◽  
K Yamaguchi ◽  
M Goto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Binding Activity ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Dna Binding Activity

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

scholarly journals Structural heterogeneity of Pseudomonas aeruginosa bacterioferritin

1994 ◽  
Vol 304 (2) ◽  
pp. 493-497 ◽  
Author(s):  
G R Moore ◽  
F H Kadir ◽  
F K al-Massad ◽  
N E Le Brun ◽  
A J Thomson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Heterogeneity ◽  
Amino Acid Sequences ◽  
Binding Study ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Binding Characteristics

The subunit composition, amino acid sequence and haem-binding characteristics of bacterioferritin (BFR) from Pseudomonas aeruginosa have been studied. Unlike other BFRs, P. aeruginosa BFR was found to contain two subunit types, designated alpha and beta, which differed considerably in their amino acid sequences. The N-terminal 69 and 55 amino acids of the alpha and beta subunits respectively were determined. The alpha subunit differed most from other BFRs. The two subunits were present in variable proportions in different preparations. The maximum stoichiometry of haem binding was found to be sample-dependent and to be different from the previously reported one per subunit [Kadir and Moore (1990) FEBS Lett. 271, 141-143]. This previous haem-binding study was shown to have been carried out with damaged protein, which contained both normal alpha and beta subunits and shorter versions of these that appeared to have been produced by cleavage of the normal subunits. The possibility that aging processes degrade ferritins and affect their haem-binding characteristics is discussed.

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