Description

Arginylendopeptidase specifically cleaves the peptide bonds at the carboxyl side of arginine residues of peptides and proteins. This enzyme is also known as mouse submaxillary protease D¹⁾ or as mouse EGF binding protein type C³⁾. This product has been treated with TLCK and TPCK to remove trace contamination of trypsin-like and chymotrypsin-like proteases activities after purification. Usually, this enzyme is used in supplied buffer under the following conditions; S/E ratio: 50 - 200, reaction temperature: 37°C, reaction time: 3 - 15 hours.

Storage

–20°C

Application

This enzyme is useful in the analysis of the primary structure of proteins and peptides.

Source

Mouse submaxillary glands

Form

Solution in 5 mM sodium phosphate buffer (pH7.2) containing 50% glycerol

Volume

0.5 mg/vial

Purity

Homogeneous on SDS-PAGE. No other proteases are detectable.

Properties

Molecular weight:	25,000 (SDS-PAGE) 21,300 (gel filtration chromatography)
Optimum pH:	8.0-9.0
Isoelectric point:	5.65
Inhibitors:	Phenylmethane sulfonyl fluoride (PMSF) Diisopropyl fluorophosphate (DFP)
Tolerance to denaturants:	Stable against	≤ 2 M urea ≤ 0.1 M guanidine-HCl ≤ 0.05% SDS
Optimum temperature:	50°C
Thermostability:	Stable below 55°C (pH7.0, 15 min.)

Supplied buffer (5 ×)

Volume :	1 ml
Component :	250 mM Sodium phosphate, pH8.0

Definition of Activity

One unit of enzyme activity corresponds to the amount required to produce 1 µmol of p-nitroaniline from benzoyl-DL-arginine p-nitroanilide in 1 minute at 37°C, pH8.0.

Substrate specificity

The -Arg-Pro- and -Arg-Glu- bonds are not cleaved with this protease. It has also been reported that when the P1 amino acid is Thr, Ala, CM-Cys, Met, Leu, Tyr or Gln, the -Arg-X- bond is not cleaved. This enzyme may cleave also -Lys-X- bonds located in basic amino acid stretches like -Lys -Lys-Ile-, -Lys-Lys-Arg-, and Arg-Lys-Lys-. The following table shows the reactivity of this enzyme against several synthetic substrates.

Substrate	Km (mM)	kcat (s-1)	kcat/Km
Benzoyl-DL-Arg-pNA	0.29	147	500
Benzoyl-DL-Lys-pNA	3.57	4.17	1.17
Tos-L-Arg-OMe	0.24	12,500	52,100
Tos-L-Lys-OMe	1.19	301	253
Benzoyl-Arg-OEt	0.13	21,700	173,000
N-Acetyl-L-Tyr-OEt		*
Glt-L-Phe-pNA		*

* Not cleaved

Note

It is necessary to denature the protein samples by chemical procedure such as carboxymethylation, prior to digestion with this enzyme.