[33] Specific modification of NH2-terminal residues by transamination

Dixon, H. B. F.; Fields, Robert

doi:10.1016/s0076-6879(72)25036-4

Cited by 92 publications

(69 citation statements)

References 26 publications

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“…Removal of N-terminal ␣-Keto Acids-␣-Keto acids can be selectively removed from the N terminus of proteins by o-phenylenediamine (28). One volume of D-proline reductase (10 g) was incubated with two volumes of sodium acetate buffer (750 mM, pH 4.5) containing 120 mM o-phenylenediamine for 17 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

Kabisch

Gräntzdörffer

Schierhorn

et al. 1999

Journal of Biological Chemistry

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Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme. The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed by o-phenylenediamine; thus, Nterminal sequencing became feasible for this subunit. L-[14 C]proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH 4 were added. Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB. prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit. The gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion. prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species.

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Section: Methodsmentioning

confidence: 99%

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

Kabisch

Gräntzdörffer

Schierhorn

et al. 1999

Journal of Biological Chemistry

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“…This latter reaction gives rise to transmination of the amino group yielding the corresponding a-keto acid. Transaminated porcine pancreatic phospholipase A2 prepared by reaction of phospholipase A2 with sodium glyoxalate [31] was found to be catalytically inert toward micellar substrates (unpublished results). Therefore the complete loss of enzymatic activity of phospholipase A2 by reaction with phenylglyoxal monohydrate is due to the accompanying transamination reaction and not to modification ofarginine residues.…”

Section: Discussionmentioning

confidence: 94%

“…From the incorporation of [7-14C]phenylglyoxal monohydrate into aBoc-~(Boc), -8-phospholipase A2 it was found that about two phenylglyoxal molecules/protein molecule were present, which turned out to be located predominantly on Arg-6. After removal of the tert-butyloxycarbonyl protecting groups in these latter two phenylglyoxal-modified proteins about [30][31][32][33][34][35][36][37][38] of their respective original enzymatic activities were recovered as determined in the egg-yolk assay. These data strongly suggest that two phenylglyoxal molecules are incorporated per protein molecule and are located predominantly on Arg-6.…”

Section: Discussionmentioning

confidence: 99%

Modification of Arginine Residues in Porcine Pancreatic Phospholipase A2

Fleer¹,

Puijk²,

Slotboom³

1981

Eur J Biochem

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Although phenylglyoxal monohydrate reacts with Arg-6 in porcine pancreatic phospholipase Az, concomittantly the a-amino group of the N-terminal Ala-1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg-6 modification. Upon reaction of [7-'4C]phenylglyoxal monohydrate with a-amino-blocked phospholipase Az analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg-6. Removal of a-amino-blocking groups after the modification reaction furnished the corresponding Arg-6-modified phospholipases possessing 30-38 "/, of their original specific enzymatic activities in the egg-yolk assay.After reaction of 1,2-[l-'4C]cyclohexanedione with porcine phospholipase A2 the crude reaction mixture was purified by chromatography on quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of ''C-labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg-6. Cyclohexanedione modification of Arg-6 in phospholipase A2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg-6 by cyclohexanedione are in agreement with previous findings that Arg-6 is involved in the interaction of the enzyme with lipid-water interfaces.Phospholipase Az has been isolated from many sources [1,2]. It is a small ( M , about 14000) water-soluble protein, catalyzing the hydrolysis of 3-sn-phosphoglycerides, whereas 1 -sn-phosphoglycerides are competitive inhibitors [3]. The enzyme has an absolute requirement of Ca" and reaches its highest activity when bound to aggregated substrates [4]. It has been shown that the snake venom enzymes bind Caz+ and substrates via an ordered mechanism in which Ca2+ is bound first [ 5 ] , while for porcine pancreatic enzyme a random mechanism has been proposed [4,6]. Investigations of the binding of pancreatic phospholipase Az to aggregated substrate and substrate analogs, have led to the proposition of a micelle-binding site on the porcine enzyme, which is topologically distinct from the active centre [7,8]. For this binding site, the term 'interface recognition site' has been proposed and indications were found for an important role of the N-terminal part of the protein in the formation of the interface recognition site [8 -101. Furthermore, it was found that the Arg-6/Ser-7 bond was protected from trypsine cleavage when porcine phospholipase AZ was bound to micelles [lo], strongly suggesting that Argd belongs to the interface recognition site. To further elaborate the role of Arg-6 in the protein, an arginine-modification study was started. Takahashi [I11 and Borders and Riordan [I21 have shown the applicability of vicinal diketons in arginine modifi...

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“…(1 977). Standard conditions for the Strecker synthesis were used for steps 4 and 5 , and usual transamination conditions (Dixon and Fields, 1972) for step 6 of the spot staining for 'phosphate' into material with about half its mobility at pH 2, about 0.7 of its mobility at pH 3.5 and slightly greater mobility at pH 6.5. The solution was stirred with a sulphonic resin (Dowex 50. hydrogen form) to decompose the excess of borohydride, and the suspension was filtered through a further portion of the same resin, and evaporated to dryness.…”

Section: -Pho\phonolr~ Trc ( I ( Idmentioning

confidence: 99%

The synthesis of 3‐phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid

Sparkes¹,

Rogers²,

Dixon³

1990

European Journal of Biochemistry

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3‐Phosphonoalanine has been made by the Strecker synthesis from phosphonoacetaldehyde, which is easily prepared from vinyl acetate. It gives phosphonopyruvate by transamination when treated with glyoxylate. Phosphonolactate, an analogue of phosphoglycerate, is prepared by reducing phosphonopyruvate. Diazotization of phosphonoalanine was investigated as a route for making phosphonolactate: addition of NaNO2 to the isoelectric form of phosphonoalanine gave much scission of the C‐P bond with release of phosphate; addition of HBr prevented this release and gave largely the bromo acid. The supplement reports the synthesis of arsonolactate, a similar analogue, by treating chlorolactate with alkaline arsenite.

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[33] Specific modification of NH2-terminal residues by transamination

Cited by 92 publications

References 26 publications

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

Identification of d-Proline Reductase fromClostridium sticklandiias a Selenoenzyme and Indications for a Catalytically Active Pyruvoyl Group Derived from a Cysteine Residue by Cleavage of a Proprotein

Modification of Arginine Residues in Porcine Pancreatic Phospholipase A2

The synthesis of 3‐phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid

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