The use of penicillocarboxypeptidase-S1 in amino acid sequencing

Hui, Anne; Rao, L.G.; Kurosky, A.; Jones, Stephen R.; Mains, Geoff; Dixon, Joan W.; Szewczuk, A; Hofmann, Theo

doi:10.1016/0003-9861(74)90434-2

Cited by 18 publications

(4 citation statements)

References 21 publications

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“…If Asp-376 is indeed the C-terminal residue of peptide Ac, then the C-terminal sequence expected would be -Pro-Ser-Asp. It has been observed (Hui et al, 1974) that penicillocarboxypeptidase S, does not readily cleave bonds between neighbouring residues with small side chains, so that the liberation only ofaspartic acid from this peptide is a finding consistent with the analysis.…”

Section: C-terminal Determinationssupporting

confidence: 78%

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

Parr¹,

Hofmann²,

Connell³

1976

Biochemical Journal

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The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.

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Section: C-terminal Determinationssupporting

confidence: 78%

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

Parr¹,

Hofmann²,

Connell³

1976

Biochemical Journal

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“…The peptides were also subjected to automated sequence studies in a Beckman 890 C sequencer (Edman, 1967). Carboxy-terminal amino acid analysis was performed on the isolated fragments using penicillocarboxypeptidase S-l in a pyridine/formate buffer at pH 3.0 for 4 h (Hui et al, 1974) at an enzyme to substrate ratio of 1:40). The digestion was stopped by the addition of 10% trichloroacetic acid, which precipitated the undigested protein.…”

Section: Methodsmentioning

confidence: 99%

Identification of membrane-embedded domains of lipophilin from human myelin

Kahan¹,

Moscarello²

1985

Biochemistry

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The organization of lipophilin in the intact human myelin membrane has been studied by labeling with the carbene photogenerated from 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). This hydrophobic probe labels mostly lipophilin (the main intrinsic protein of myelin) and the lipids within the bilayer. The domains of lipophilin which are embedded within the membrane have been identified by proteolytic fragmentation of the [125I]TID-labeled myelin, extraction with organic solvents, and separation by chromatography. Four labeled peptides were purified in this way. Polyacrylamide gel electrophoresis, amino acid compositions, automated sequencing, and carboxy-terminal analyses identified a 15K molecular weight peptide, T1 (residues 1-143), as representing the amino-terminal fragment, a 10K peptide, T2 (residues 1-97), representing a smaller amino-terminal fragment, a 5K peptide, T4 (residues 53-97), which represented the COOH-terminal half of peptide T2, and a 7K peptide, T3 (residues 205-268), which represented a sequence near the COOH terminus of lipophilin. The specific radioactivities of the peptides were determined; peptides T1 and T2 had similar specific activities, which were twice the specific activities of peptides T3 and T4. The data provide direct chemical evidence that human lipophilin has membrane-embedded domains between residues 1-97, 53-97, and 205-268, in agreement with some of the predictions of other investigators based on the sequence of bovine myelin lipophilin (proteolipid apoprotein) and a hydrophobicity diagram.

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“…C-Terminal residues were determined after digestion with penicillocarboxypeptidase S1 (Hui et al, 1974) in 0.3ml of 0.03M-pyridine/acetate buffer, pH4.7, at 37'C with enzyme/substrate ratios of about 1: 100 (mol/mol). Digestionwasstopped byimmersing the tubes in boiling water for 5min.…”

Section: C-terminal Determinationmentioning

confidence: 99%

Fb′2, a new peptic fragment of human immunoglobulin G

Parr

Connell

Kells

et al. 1976

Biochemical Journal

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The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb'2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb's was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb'2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb's; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb'2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.

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The use of penicillocarboxypeptidase-S1 in amino acid sequencing

Cited by 18 publications

References 21 publications

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

Identification of membrane-embedded domains of lipophilin from human myelin

Fb′2, a new peptic fragment of human immunoglobulin G

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