Solid‐Phase Methods in Protein Sequence Analysis

Laursen, Richard A.; Machleidt, Werner

doi:10.1002/9780470110461.ch6

Cited by 47 publications

(1 citation statement)

References 158 publications

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“…The amino acid sequencing 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 the procedure of Laursen (1 966) and Laursen and Machleidt (1980). In brief, 30-pl aliquots of the phosphopeptide peak fractions resolved from HPLC were incubated with the reaction membrane at 56°C for 20 rnin and then washed with methanol and deionized water.…”

Section: Amino Acid Sequence Analysis and Determination Of Phosphorylmentioning

confidence: 99%

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Yang

1994

Journal of Neurochemistry

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In a previous study, protein kinase FJglycogen synthase kinase-3 (F,/GSK-3) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase F,/GSK-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase F,/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C,8 reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thrg7-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase FJGSK-3, implicating a physiologically relevant role of F,/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVTg4(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [32P]MBP phosphorylated by kinase FJGSK-3, further indicating that kinase FJ GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin. Key Words: Myelin basic protein-Protein kinase F,/glycogen synthase kinase-3-Phosphorylation site-Consensus sequence motif. J. Neurochern. 62, 1596-1 603 (1 994).

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Section: Amino Acid Sequence Analysis and Determination Of Phosphorylmentioning

confidence: 99%

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Yang

1994

Journal of Neurochemistry

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High‐Performance Liquid Chromatography: Analytic and Preparative Applications in Protein‐Structure Determination

Hughes

Wilson

1983

Methods of Biochemical Analysis

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Edman Degradation

2010

Comprehensive Organic Name Reactions and Reagents

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The reaction between phenyl isothiocyanate and the free amino group of the N ‐terminal amino acid within a polypeptide to generate a thiourea, which upon the intramolecular cyclization via the nucleophilic addition of the amino group to adjacent amide carbonyl cleaves the N ‐terminal amino acids as a phenylthiohydantoin derivative and leaves the second amino acid as the N ‐terminus again, is generally known as the Edman degradation. This method shows a few disadvantages and, to overcome these weaknesses, the reaction has been carried out by means of solid‐phase support synthesis (SPSS). Further development has made this degradation valuable for sequencing the C‐terminal segments of hydroxyethylamine‐derived peptides and a useful method for the synthesis of 2‐iminohydantoins.

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Solid‐Phase Methods in Protein Sequence Analysis

Cited by 47 publications

References 158 publications

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

High‐Performance Liquid Chromatography: Analytic and Preparative Applications in Protein‐Structure Determination

Edman Degradation

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Solid‐Phase Methods in Protein Sequence Analysis

Cited by 47 publications

References 158 publications

Protein Kinase FA/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr97‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Protein Kinase FA/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr97‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

High‐Performance Liquid Chromatography: Analytic and Preparative Applications in Protein‐Structure Determination

Edman Degradation

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Product

Resources

About

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs

Protein Kinase F_A/Glycogen Synthase Kinase‐3 Predominantly Phosphorylates the In Vivo Site Thr⁹⁷‐Pro in Brain Myelin Basic Protein: Evidence for Thr‐Pro and Ser‐Arg‐X‐X‐Ser as Consensus Sequence Motifs