Two-dimensional zymography in detection of proteolytic enzymes in wheat leaves

Małgorzata Grudkowska,Piotr Lisik,Krystyna Rybka

doi:10.1007/s11738-013-1371-1

Abstract

Proteolytic enzymes in wheat leaves were studied using zymographic detection of enzyme activities on one-way (1D) SDS-polyacrylamide gels and two-dimensional (2D) ones, on which protein samples were isoelectrofocused prior to PAGE separation. Gelatin of concentration 0.1 %, copolymerized into SDS-PAGE gels, digested by active proteinases enabled detection of those enzymes. On 1D gels, seven bands were seen and assigned to particular families through the use of specific inhibitors. Metalloproteinases inhibited by 20 mM EDTA were detected as 150 kDa band; aspartic proteinases were assigned to 115–118 kDa double band by using 25 mM pepstatin; 10 mM phenylmethylsulfonyl fluoride used for detection of serine proteinases pointed to band of 70 kDa and finally due to 10 μM E-64 inhibitor, cysteine proteinases of 37 and 40 kDa were detected. On 2D gels, additional separation according to protein isoelectric points enabled detection of proteinase isoforms. In the range of 4.5–6 pI, six metalloproteinases as well as ten aspartic proteinases were visible, ten serine- isoforms of pI 4.5–6.8 and four cysteine proteinases of 4.5–5.0 pI were found. Presented results were detected as reproducible results observed at least in four independent biological replications.

Highlights

Zymography is a widely accepted analytical technique used to detect enzymes in tissue extracts
Metalloproteinases inhibited by 20 mM EDTA were detected as 150 kDa band; aspartic proteinases were assigned to 115–118 kDa double band by using 25 mM pepstatin; 10 mM phenylmethylsulfonyl fluoride used for detection of serine proteinases pointed to band of 70 kDa and due to 10 lM E-64 inhibitor, cysteine proteinases of 37 and 40 kDa were detected
We report the advantage of using two-dimensional (2D) zymography in combination with specific proteinase inhibitors to reveal the pattern of major leaf proteases in wheat

Summary

Introduction

Zymography is a widely accepted analytical technique used to detect enzymes in tissue extracts. Acta Physiol Plant (2013) 35:3477–3482 renaturation is followed by substrate digestion (Zhang and Jones 1995) All of these are carried out in the gel soaked in a buffer of pH appropriate to the pH optimum of studied enzyme. A modification of this method is the detection of proteinase inhibitors in the gel While such a gel is incubated in a buffer containing the enzyme of interest nearly the whole substrate incorporated into the gel is digested beside the places where enzyme inhibitors are present. Conditions of extraction and electrophoresis may influence the reduction of enzyme activities leading to underestimation of their amount (Pan et al 2011) In this short communication, we report the advantage of using two-dimensional (2D) zymography in combination with specific proteinase inhibitors to reveal the pattern of major leaf proteases in wheat. We demonstrate that zymography is still the technique of choice in protease studies and permits very good separation of distinct proteinase families

Materials and methods

Results and discussion

E-64 EP1 EP2 EP3 EP4 EP5