Endopeptidases lysyl endopeptidase

KINETIC ISOTOPE EFFECT LYSYL ENDOPEPTIDASE LYSYL HYDROXYLASE LYSYL OXIDASE Lysyl residue,... 

It is important to keep the F(ab )2-carbohydrazide, once removed from the 2.5 M carbohydrazide soln, in the presence of a large excess of Trasylol. If this is not done, the coupling is rapidly reversed by remaining traces of lysyl endopeptidase. 

Proteolysis of peptides and proteins by enzymes occurs in a selective or nonselective manner. Chymotrypsin, trypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease, and en-dopeptidase Asp-N are frequently listed as selective enzymes, whereas thermolysin, pepsin, subtilisin, and elastase belong to the nonselective enzymes, although thermolysin preferentially cleaves peptide bonds before hydrophobic residues. 

Scheme 8 Formation of Two Interchain Disulfide Bonds with Different Connectivities Using Two Kinds of Protecting Groups for the Cysteine Residues and Activation via the Npys Derivative. Synthetic Schemes for the Two Isomers of the ro-Conotoxin MVIIC Fragment Obtained by Digestion with Lysyl Endopeptidase and Thermolysin19 ...

Table 1 Eight Possible Lysyl Endopeptidase Fragments of p-Conotoxin GIIIB with Different Disulfide Connectivities and Ratios of (PTH-Cys)2 during Their Sequencing 72 "...

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

Cucumis melo (oriental pickling melon) (Cucurbitaceae) [seedl Cucumis CMCTI-I (28 aa 3.1 kDa 6 Cys) Trypsin [127 pM], Lysyl endopeptidase [207 pM] (K4-I5) [539]... 

After removing, the last wash, the gel pieces were brought to dryness in a Speedvac and then hydrated by adding 1 tl/mm (initial estimated gel volume) of a freshly prepared enzyme solution made by mixing one volume 0.1 mg/ml trypsin (Promega modified) or lysyl endopeptidase (Wako) with two volumes 200 mM NH4HCO3 If necessary, additional enzyme solution (0.0333 mg/ml) was added to totally immerse the gel pieces. 

Figure 1. Reverse phase HPLC separation of tryptic (A, B) and lysyl endopeptidase (C, D) in gel digests of 25 pmol aliquots of human transferrin. Following SDS-PAGE, the gel was stained with Coomassie Blue and the bands of interest were then excised, digested and subjected to HPLC as described m Materials and Methods. The digests shown in the top two chromatograms were carried out as described in Materials and Methods while the digests shown in the bottom two chromatograms were carried out in the presence of 0.02% Tween 20. All four digests were chromatographed at 50 /il/min on a 1 x 250 mm Vydac C-18 column.

Lysyl endopeptidase/h-insulina Achromobacter lyticus Nature 280 412(1979)... 

Clostripain (EC 3.4.22.8) an SH-dependent, liyp-sin-like protease (M, 50,000) with endopeptidase and amidase-esterase activity, isolated from culture filtrates of Clostridium histolyticum. Ihe endopeptidase acivity hydrolyses proteins, while the amidase-esterase activity cleaves synthetic amino acid amides and amino acid esters. It attacks only arginyl and lysyl re-adues, and is therefore used to isolate large peptide fragments without prior chemical modification of the sutetrate. 

A group of heritable disorders. Clinical features include hyperelastic skin, hyperextensible joints, easy bruising and poor wound healing. Collagen has decreased content of hydroxylysine. Deficient enzyme may be Lysyl oxidase (EC 1.4.3.6), Lysyl protocollagen hydroxylase (EC 1.14.11.4) or Procollagen peptidase (C-endopeptidase EC 3.4.24.19, or A-endopepti-dase EC 3.4.24.14). 

An epidermal growth factor (EOF) was treated with lysyl endopeptidase to produce des-B30 insulin. These compounds were separated on a C]g column (A = 220nm) using a 35-min 75/25 ->40/60 water/acetonitrile with 1% TFA gradient [1312], EGF eluted in roughly 16 min and insulin in 10 min. Peak shapes were excellent. 

Figure 8 (A) Separation of the lysyl-C endopeptidase digest of calreticulin by packed capillary HPLC and detection of its eluate with ESI/TSQMS. The pyridyl-ethylated calreticulin was digested with lysyl-C endopeptidase and applied to a capillary column (1.0mm IDxIOcm) packed with CapcellPak C18 (3 urn). The peptides are eluted at a flow rate of 50 nL/min with a linear gradient of increasing concentration from 0 to 100% of acetonitrile containing 0.1 % TFA. The eluent from the capillary column was detected with ESI/TSQMS. (B) Heterogeneity in molecular weight of the L-16 glycopeptide derived from calreticulin observed by the packed capillary HPLC-ESI/TSQMS analysis. The spectrum shown is one decon-voluted from the original mass spectrum.

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