The Determination of the Order of Lysine‐containing

Perham, Richard N.; Jones, G. M. Thelwall

doi:10.1111/j.1432-1033.1967.tb00110.x

Cited by 51 publications

(12 citation statements)

References 18 publications

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“…The fact that all basic amino acid residues but one are clustered toward the carboxyl-terminal region of the molecule of pepsin has been noted before [28,29]. The amino acid sequence of the C-terminal27-residue portion of fragment CB-1 is in agreement with amino acid sequences reported for this region before [28-301. The data presented in this paper do not fully agree with the partial structure proposed for the carboxylterminal fragment of pepsin by Ostoslavskaya and co-workers [31].…”

Section: Discussionsupporting

confidence: 79%

“…The similarity in amino acid sequences of the carboxyl-terminal portions of hog pepsin and calf rennin [32] has been pointed out earlier [28]. While there is a remarkable homology between the amino acid sequences of the 28-residue carboxyl-terminal regions of these two proteins, there is practically no similarity in amino acid sequences preceding the tryptophan residue (cf.…”

Section: Discussionmentioning

confidence: 99%

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Amino Acid Sequence of C‐Terminal Fragment of Hog Pepsin

Kostka

Morávek

Šorm

1970

European Journal of Biochemistry

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The C-terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37-residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. I n parallel experiments the sequence of seventeen amino acid residues in the N-terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C-terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp-Val-Pro-Thr-Ser-Ser-Gly-GluLeu-Trp-Ile-Leu-Gly-Asp-Val-Phe-Ile-Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-LysVal-Gly-Leu-Ala-Pro-Val-Ala. These results extend and partly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Amino Acid Sequence of C‐Terminal Fragment of Hog Pepsin

Kostka

Morávek

Šorm

1970

European Journal of Biochemistry

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“…The buffers (pH 6.5 and 3.5) were those described by Perham [20]. The peptides containing c-pyridoxyllysine were located on paper because of their blue fluorescence under ultraviolet light.…”

Section: Paper Electrophoresismentioning

confidence: 99%

Identification of Lysines Reactive with Pyridoxal 5′‐Phosphate in Glyceraldehyde‐3‐phosphate Dehydrogenase

Forcina¹,

Ferri²,

Zapponi³

et al. 1971

European Journal of Biochemistry

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Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase is almost completely inhibited by reaction of 4 moles of pyridoxal 5'-phosphate per mole of subunit chain of holoenzyme.Reduction of the pyridoxal 5'-phosphate inactivated holoenzyme with NaBH, followed by carboxymethylation of thiol groups, and subsequent tryptic digestion, allowed the separation of two strongly fluorescent peptides. These have been identitied as peptides T19 + T20 (residues 184 to 192) and peptides T22 + T23 (residues 198 to 216) of the sequence published by Harris and Perham for porcine enzyme, in which lysines 191 and 212 are blocked by pyridoxal 5'-phosphate. Evidence for other lysyl residues reacting to a lesser extent with the reagent is reported: among such residues, only lysine-2 (contained in the N-terminal peptide) has been unambigously identified. No reaction takes place at lysine-183. The homology between the sequence around lysine-212 and the sequence around the essential lysine residue in glutamate dehydrogenase is stressed. The role of pyridoxal5'-phosphate reactive lysines in the mechanism of glyceraldehyde-3-phosphate dehydrogenase is discussed.A considerable amount of evidence is available, at present, on the participation of lysyl residues in the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase.In the course of the enzyme-catalyzed hydrolysis of p-nitrophenylacetate or acetyl phosphate, the NAD free protein isolated from a variety of sources can be N-acetylated at a specific lysyl residue [1,2], with a concomitant loss of activity in the oxidative phosphorylation of the natural substrate 3-phosphoglyceraldehyde [ 1,3]. The unique lysine acetylated has been shown to be lysine-183 in the sequence of the pig [4], rabbit [2], lobster [5] and yeast [6] enzymes and to be spatially close to the active thiol cysteine-149 in the three dimensional structure of the tetrameric apoenzyme.Moreover, previous results from this laboratory [7] had shown that glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscle is reversibly inactivated by low concentrations of pyridoxal 5'-phosphate, by now a well established reagent for studying the role oflysyl &-amino groups in the activity o f a number of enzymes [8,9,10]. Since lysine-183 is reactive only in the NAD-free enzyme, while the study with pyridoxal 5'-phosphate as a chemical modifying reagent was carried out on the NAD. protein complex, it was of interest to identify the lysines reactive with pyridoxal 5'-phosphate in the Enzyme. Glyceraldehyde-3-phosphate : NAD-oxidoreductase (phosphorylating) (EC 1.2.1.12).primary structure of the native holoenzyme. It is well known, in fact, that the binding of NAD causes a conformational change in the enzyme [ll], and that the coenzyme may act as an active site director in glyceraldehyde-3-phosphate dehydrogenase modification [12].The results reported in this paper show that lysines-I91 and 212 in the sequence published by Harris and Perham for porcine glyceraldehyde-3-phosphate dehydrogenase [I31 are the most reactive with...

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“…Enzymic digestion and cyanogen bromide cleavage, amino acid analysis, and N-terminal analysis by the dansyl technique were carried out as previously described [ 1 ]. Separation of peptides by paper electrophoresis and chromatography was performed according to Perham and Jones [7] and dansyl-Edman degradation of peptides was carried out essentially as described by Gray and Hartley [8]. Amide assignments in peptides were made from the electrophoretic mobility (m) at pH 6.5 [9], defining the mobility of aspartic acid as -1.00.…”

Section: Methodsmentioning

confidence: 99%