Cleavage of glucagon by α‐ and β‐trypsin

Keil-Dlouhá, V.; Zylber, N.; Tong, Nguyen Thanh; Keil, B.

doi:10.1016/0014-5793(71)80372-1

Cited by 33 publications

(23 citation statements)

References 31 publications

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“…3a) and in tissue sections (Ulrich et al 1987). Since trypsin cleaves at the C-terminus of lysine (Keil-Dlouha et al 1971;Olsen et al 2004), the possibility exists that the N-terminal lysine may be important for RT-97 IR in addition to lysine in the C-terminal side of the peptide. To test this hypothesis, we generated phospho-variants of wild type (KSP) and mutant glycine serine proline (GSP) peptides derived from the mouse NF-H sequence, in which glycine replaced lysine in the KSP motifs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Neurofilament tail phosphorylation: identity of the RT‐97 phosphoepitope and regulation in neurons by cross‐talk among proline‐directed kinases

Veeranna

Lee

Pareek

et al. 2008

Journal of Neurochemistry

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As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C‐terminal tails, which is recognized by the monoclonal antibody, RT‐97. Here, we demonstrate in vivo that RT‐97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT‐97 epitope during early postnatal brain development. Purified ERK1,2 generated RT‐97 on both KSP motifs on recombinant NF‐H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT‐97 epitope generation in primary hippocampal neurons was regulated by extensive cross‐talk among ERK1,2, c‐Jun N‐terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT‐97 generation. Cdk5 influenced RT‐97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT‐97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT‐97 IR by activating ERK1,2. Thus, cdk5 influences RT‐97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.

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Section: Resultsmentioning

confidence: 99%

Neurofilament tail phosphorylation: identity of the RT‐97 phosphoepitope and regulation in neurons by cross‐talk among proline‐directed kinases

Veeranna

Lee

Pareek

et al. 2008

Journal of Neurochemistry

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“…We observed by MALDI-TOF MS analysis of the tryptic or combined trypsin/endo-GluC digests that the analogs photoreactive at position 4, 5, 6, 9, or 10 are cleaved by the chymotrypsin-like activity derived from trypsin (from Sigma) as previously described (data not shown) (6,7,14). This cleavage even occurs with the sequencing-grade modified trypsin (from Promega) that should be extremely resistant to autolysis.…”

Section: Resultsmentioning

confidence: 99%

CγH2 of Met174 Side Chain Is the Site of Covalent Attachment of a Substance P Analog Photoactivable in Position 5

Sachon

Bolbach

Chassaing

et al. 2002

Journal of Biological Chemistry

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Analogs of substance P (H-RPKPQQFFGLM-NHThe substance P (H-RPKPQQFFGLM-NH 2 ) NK-1 receptor is a member of the class I family within the superfamily of Gprotein-coupled receptors. The NK-1 receptor has been cloned in several species, among them the murine, the rat, and the human. The hNK-1 1 receptor is composed of 407 amino acids organized in 7 membrane-spanning segments. As for all other receptors from this superfamily, the molecular basis of the binding of the preferred endogenous ligand SP to the NK-1 receptor is far from being well understood. Structure-activity relationships in SP (amino acid substitutions or modifications) (1) as well as in the NK-1 receptor (site-directed mutagenesis, construction of deleted or chimeric receptors) (2) have been studied extensively. Beside these studies, a complementary and more direct approach to identify the key points in the interaction between a peptide and a protein is photoaffinity labeling (3).Several photoaffinity-labeling studies of the NK-1 receptor have already been reported (see Fig. 3 To define further the binding site for SP within the NK-1 receptor, we have synthesized photoreactive analogs, [BAPA 0 , (p-Bzl)Phe x , Met(O 2 ) 11 )]SP, incorporating a biotinyl sulfone amino pentanoic acid (BAPA) derivative at the N terminus and the photoreactive amino acid (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence of SP. We previously showed that the analog modified at position 7 has no affinity (M) for the NK-1 receptor (6). The peptide analogs of SP photoreactive at position 4, 5, 6, 9, or 10 have high affinity for the two binding sites associated with the NK-1 receptor transfected in chinese hamster ovary cells and are potent agonists in activating both phosphatidylinositol hydrolysis and cAMP accumulation (11).In this study, we show that these photoreactive analogs also give good yields of photolabeling of the NK-1 receptor transfected in chinese hamster ovary cells. We have used a methodology of photolabeling already described to identify the site of photoinsertion of SP analogs photoactivable at position 8, in the NK-1 receptor (6, 7). This methodology is based on the use of streptavidin-coated magnetic beads to purify the covalent ligand/receptor complex after enzymatic and/or chemical digestions and subsequent analysis by MALDI-TOF MS to identify the site of photoinsertion within the receptor (6, 7). EXPERIMENTAL PROCEDURES Materials

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“…In a parallei study [3] we have shown, that pure cu-trypsin cleaves in glucagon exclusively the bonds adjacent to three basic amino acid residues. The presence of ar-trypsin, still surviving the treatment of the autolysate with TLCK, could therefore explain the cleavage at these sites, but not the degradation at the aromatic amino acid residues.…”

Section: Discussionmentioning

confidence: 95%

“…Recoveries of alI isolated peptides are compared with the results of previous studies [3,8,91 in table 1.…”

Section: Incubation Of Glucagon With $-Trypsin and Tlckwiypsin And Anmentioning

confidence: 99%

Proteolytic activity of pseudotrypsin

et al. 1971

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Cleavage of glucagon by α‐ and β‐trypsin

Cited by 33 publications

References 31 publications

Neurofilament tail phosphorylation: identity of the RT‐97 phosphoepitope and regulation in neurons by cross‐talk among proline‐directed kinases

Neurofilament tail phosphorylation: identity of the RT‐97 phosphoepitope and regulation in neurons by cross‐talk among proline‐directed kinases

CγH2 of Met174 Side Chain Is the Site of Covalent Attachment of a Substance P Analog Photoactivable in Position 5

Proteolytic activity of pseudotrypsin

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