Relatedness among proteins: A new method of estimation and its application to immunoglobulins

Marchalonis, John J.; Weltman, Joel K.

doi:10.1016/0305-0491(71)90316-6

Cited by 155 publications

(95 citation statements)

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“…We therefore calculated SAQ values (24) for the pairwise comparison of the P. cepacia dienelactone hydrolase with other dienelactone hydrolases for which the amino acid composition could be predicted from the nucleotide sequence (17,39,50). We also compared the enzyme with 3-oxoadipate enollactone hydrolases for which composition data had been reported (25,34).…”

Section: Discussionmentioning

confidence: 99%

Dienelactone hydrolase from Pseudomonas cepacia

et al. 1993

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(15,16,42,46,49). As a result of chloride elimination, the product, a cis-or trans-dienelactone (4-carboxymethylenebut-2-en-4-olide), carries an additional double bond as compared with muconolactone, the cycloisomerization product of unsubstituted cis,cis-muconate (Fig. 1). While muconolactone first must be converted to 3-oxoadipate enol-lactone to make lactone hydrolysis possible, the dienelactones already have an enol-lactone structure and can be hydrolyzed directly.Despite the overall structural similarity between 3-oxoadipate enol-lactone and the dienelactones, they are converted by different enzymes in Pseudomonas sp. strain B13 (46). The 3-oxoadipate enol-lactone hydrolase (EC 3.1.1.24) of this strain does not attack the dienelactones, whereas the dienelactone hydrolase converts both dienelactone isomers but not 3-oxoadipate enol-lactone. The B13 dienelactone hydrolase, which appears to be identical to the corresponding pAC27-encoded enzyme (17), was further characterized and found to resemble Pseudomonas putida 3-oxoadipate enol-lactone hydrolase with respect to molecular mass, inhibition by p-chloromercuribenzoate, amino acid composition, and NH2-terminal amino acid sequence (29). An identical pentapeptide, neighboring a cysteine residue in both hydrolases (17), was interpreted to represent a region conserved during divergent evolution of the hydrolases because of its proximity to an active-site thiol. However, detailed X-ray crystallographic studies of the pAC27-encoded dienelactone hydrolase suggested that Cys-123 rather * Corresponding author. than Cys-60, which neighbors the pentapeptide, is involved in lactone hydrolysis (35)(36)(37). This leaves the relationship between Pseudomonas sp. strain B13 dienelactone hydrolase and P. putida 3-oxoadipate enol-lactone hydrolase open to question.Recently, the investigation of enzymes induced during growth of several Alcaligenes strains and Pseudomonas cepacia with 4-fluorobenzoate demonstrated the existence of dienelactone hydrolases with quite different substrate specificities (45). Like the enzyme from Pseudomonas sp. strain B13, the pJP4-encoded dienelactone hydrolase of the 2,4-dichlorophenoxyacetate-utilizing strain Alcaligenes eutrophus JMP134 hydrolyzes both dienelactone isomers. These two enzymes, termed type III dienelactone hydrolases, are closely related to each other as indicated by 54.2% amino acid sequence identity between the pAC27-and pJP4-encoded hydrolases (39). The substrate specificity of another closely related protein, TcbE of Pseudomonas sp. strain P51 (50), is not yet known. In comparison, the dienelactone hydrolases (type I) of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes sp. strain A7 convert the trans-dienelactone much faster than the cis isomer (45). A dienelactone hydrolase (type II) with opposite specificity was induced by P. cepacia. We now report on the purification of the 3-oxoadipate enol-lactone hydrolase and the dienelactone hydrolase of P. cepacia and present some properties of the latter enzym...

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Section: Discussionmentioning

confidence: 99%

Dienelactone hydrolase from Pseudomonas cepacia

et al. 1993

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“…Enzyme samples dialysed against distilled water were evaporated to dryness and hydrolysed in 6 M HC1 containing 0.1 % (v/v) phenol for 24 and 48 h. Amino acid composition was determined on a Waters Pico-Tag system using 2-aminohexanoic acid (norleucine) as an internal standard. Compositional relatedness between PCs was estimated using the procedure of Marchelonis & Weltman (1971). N-terminal sequencing was performed using the automated Edman degradation procedure.…”

Section: Purification Of Pc By Dye-ligand Affinity Chromatographymentioning

confidence: 99%

Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography

Modak

Kelly

1995

Microbiology

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Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-'. In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-'. The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140000 Da and was absolutely dependent on acetyl-CoA for activity. AcetylCoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.

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“…The similarity between the amino acid composition of the purified a and 13 subunits of rice glutelin and the corresponding subunits of other 11S seed proteins was determined as described (6,16 (6). When SAQ is equal to or less than the critical value, there is high probability that the proteins have similar amino acid sequences (6,16).…”

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confidence: 99%

Biochemical Characterization of Rice Glutelin

Teng¹,

Luthe²

1985

Plant Physiol.

110

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ABSTRACI MATERIALS AND METHODSThe two major subunits of rice glutelin, the acidic (a) and basic (f) (1,2,5,7,10,23,24,26); both the large and small subunits consist of polypeptides exhibiting acidic and basic isoelectric points, respectively (4,8,24); and the amino acid compositions of oat globulin and soybean legumin (glycinin) appear to be quite similar (4). Although it is classified as a glutelin because it is insoluble in salt solutions, the major storage protein fraction of rice has some characteristics which indicate that it is similar to the 'legumin-like' protein of oat and soybeans. The two major subunits of glutelin have mol wt near 40 and 20 kD (13,15,29), and rice glutelin is synthesized as a precursor polypeptide which is posttranslationally cleaved to form two smaller subunits (15,29). Like oat globulin (1), rice glutelin appears to be preferentially synthesized on membranebound polysomes (15,29) Most of the chemicals were purchased from Sigma Chemical Company and Fisher Scientific, except that acrylamide, N,N'-methylene-bis-acrylamide, SDS, and TEMED2 were from BioRad, ampholines were from LKB, and polybuffer exchangers were from Pharmacia Fine Chemicals.Rice Protein Extraction. Dehulled rice seeds were ground into flour using a blender. The rice flour was then defatted by stirring with petroleum ether for 1 h at room temperature, filtered through a Buchner funnel, and air-dried overnight. Rice proteins, albumin, globulin, prolamin, and glutelin were sequentially extracted from defatted rice flour as described (15). The glutelincontaining supernatant was dialyzed against distilled H20 at 4C, centrifuged at 4000g for 30 min, lyophilized, and alkylated with 4-vinylpyridine (4).Mol wt of polypeptides of rice glutelin were estimated by SDS-PAGE using a 5 to 20% (w/v) acrylamide gradient gel as described previously (4, 14).Glutelin Acidic (a) Subunit Purification. Glutelin a subunit was purified by chromatofocusing using modifications of the manufacturer's (Pharmacia) procedure. The matrix was Polybuffer exchanger PBE 94 (Pharmacia). The column (1.0 x 40 cm) was equilibrated with about 20 column volumes of start buffer containing 9 M urea, 25 mm Tris-acetate (pH 8.3). Because of the insolubility of glutelin, it was essential to keep 9 M urea in the column buffers. Alkylated glutelin (129.72 mg) in 10 ml of start buffer was loaded onto the column. The column was then eluted with 12.5 volumes of a solution containing 30% (v/v) of 1:10 diluted Polybuffer 96 (Pharmacia; pH range, 9-6), 70% (v/ v) of 1:10 diluted Polybuffer 74 (Pharmacia; pH range, 7-4) in 9 M urea. The pH of the Polybuffer mixture was adjusted to 5.0 with acetic acid prior to the elution of the column. Fractions (2 ml) were collected and the A at 280 nm was measured using a Perkin-Elmer 552 recording spectrophotometer. SDS-PAGE was used to determine the protein composition of the fractions. Prior to loading on the gel, selected fractions (20-30 Ml) were mixed with an equal volume of 2x sample buffer (14) and boiled for 4 min.

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Relatedness among proteins: A new method of estimation and its application to immunoglobulins

Cited by 155 publications

References 20 publications

Dienelactone hydrolase from Pseudomonas cepacia

Dienelactone hydrolase from Pseudomonas cepacia

Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography

Biochemical Characterization of Rice Glutelin

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