A Solid-State Edman Degradation

Laursen, Richard A.

doi:10.1021/ja00974a069

Cited by 75 publications

(16 citation statements)

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“…Radiosequencing of the phosphorylation site of the phosphopeptides isolated from tryptic digests of 32p-tau as described above was performed by sequential manual Edman degradation essentially according to Laursen (1966) and Laursen and Machleidt (1980). In brief, the 32p-phosphopeptide fractions eluted from TLC plates and resuspended in 30% acetonitrile were coupled with reaction membrane at 56~ for 20 min and then washed in methanol and deionized water.…”

Section: Amino Acid Sequence Analysis and Determination Of Phosphorylmentioning

confidence: 99%

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Song

Yang

1995

J Protein Chem

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Previously, tau protein kinase I/glycogen synthase kinase-3 beta/kinase FA(TPKI/GSK-3 beta/FA) was identified as a brain microtubule-associated tau kinase possibly involved in the Alzheimer disease-like phosphorylation of tau. In this report, we find that the TPKI/GSK-3 beta/FA can be stimulated to phosphorylate brain tau up to 8.5 mol of phosphates per mol of protein by heparin, a polyanion compound. Tryptic digestion of 32P-labeled tau followed by high-performance liquid chromatography and high-voltage electrophoresis/thin-layer chromatography reveals 12 phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and peptide sequence analysis further reveals that TPKI/GSK-3 beta/FA after heparin potentiation phosphorylates tau on sites of Ser199, Thr231, Ser235, Ser262, Ser396, and Ser400, which are potential sites abnormally phosphorylated in Alzheimer tau and potent sites responsible for reducing microtubule binding possibly involved in neuronal degeneration. The results provide initial evidence that TPKI/GSK-3 beta/FA after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau and neuronal degeneration in Alzheimer disease brains.

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Section: Amino Acid Sequence Analysis and Determination Of Phosphorylmentioning

confidence: 99%

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Song

Yang

1995

J Protein Chem

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“…Since the introduction of the solid phase approach to 5 8 N-terminal protein sequencing by Laursen (1966), several different types of functionalized supports have been described for the covalent immobilization of polypeptide samples. These include polystyrene resins, polyacrylamide resins, and glass beads substituted with aminoalkyl or aminophenyl groups (Laursen & Machleidt, 1980).…”

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confidence: 99%

Carboxylic acid‐modified polyethylene: A novel support for the covalent immobilization of polypeptides for C‐terminal sequencing

1992

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We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,l'-carbonyldiimidazole (CDI), l-ethyl-3-(3-dimethylaminopropy1)carbodiimide hydrochloride (EDC), benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly through the a-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the €-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 "C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 X 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively,

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“…The amino acid sequence analysis of the phosphopeptides isolated from tryptic digests of 32p-MBP as described above was performed on a MilliGen/Bioresearch model 6600 sequencer and the phosphorylation site was determined by sequential manual Edman degradation essentially according to Laursen (1966) and Laursen and Machleidt (1980). Briefly, 30-/xl aliquots of the phosphopeptide peak fractions resolved from HPLC were incubated with reaction membrane at 56~ for 20 min and then washed with methanol and deionized water.…”

Section: Amino Acid Sequence Analysis and Determination Of Phosphorylmentioning

confidence: 99%

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

Yang

Huang

1994

J Protein Chem

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In a previous report [Yang et al., (1987a), J. Biol Chem. 262, 7034-7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C18-reversed phase high-performance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio approximately 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio approximately 0.9) and TT(p)HYGSLPQK (molar ratio approximately 0.8) as the major phosphorylation site sequences in 32P-MBP phosphorylated by autokinase, further indicating that -Arg-X-Ser/Thr-(neutral amino acid)3-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)2-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/multifunctional protein kinase in the brain.

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A Solid-State Edman Degradation

Cited by 75 publications

References 0 publications

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Tau protein kinase I/GSK-3Β/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain

Carboxylic acid‐modified polyethylene: A novel support for the covalent immobilization of polypeptides for C‐terminal sequencing

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

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