Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

Srivastava, Ashish Kumar; Nair, J.P.; Bendigeri, Dipti; Vijaykumar, A.G.; Ramaswamy, N.K.; D’Souza, S.F.

doi:10.1007/s11738-008-0219-6

Cited by 6 publications

(3 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The over-expression of CND41 reduced Rubisco in senescent tobacco leaves whereas CND41 -silenced lines delayed senescence and maintained higher levels of Rubisco in old leaves ( Kato et al , 2005 ). Besides, activities of other chloroplast proteases have been shown to increase under senescence, as in the case of an alkaline serine-protease (subtilase) induced in leaves of spinach under salinity stress and in desiccated leaves of Ramonda serbica ( Srivastava et al. , 2009 , Kidric et al.…”

Section: Interplay Between Proteases Protease Inhibitors and Target mentioning

confidence: 99%

Plant senescence and proteolysis: two processes with one destiny

et al. 2016

View full text Add to dashboard Cite

Senescence-associated proteolysis in plants is a complex and controlled process, essential for mobilization of nutrients from old or stressed tissues, mainly leaves, to growing or sink organs. Protein breakdown in senescing leaves involves many plastidial and nuclear proteases, regulators, different subcellular locations and dynamic protein traffic to ensure the complete transformation of proteins of high molecular weight into transportable and useful hydrolysed products. Protease activities are strictly regulated by specific inhibitors and through the activation of zymogens to develop their proteolytic activity at the right place and at the proper time. All these events associated with senescence have deep effects on the relocation of nutrients and as a consequence, on grain quality and crop yield. Thus, it can be considered that nutrient recycling is the common destiny of two processes, plant senescence and, proteolysis. This review article covers the most recent findings about leaf senescence features mediated by abiotic and biotic stresses as well as the participants and steps required in this physiological process, paying special attention to C1A cysteine proteases, their specific inhibitors, known as cystatins, and their potential targets, particularly the chloroplastic proteins as source for nitrogen recycling.

show abstract

Section: Interplay Between Proteases Protease Inhibitors and Target mentioning

confidence: 99%

Plant senescence and proteolysis: two processes with one destiny

et al. 2016

View full text Add to dashboard Cite

show abstract

“…In spinach chloroplasts, two subunits of alkaline serine proteinase characterized by molecular weights 63 and 32 kDa and maximal activity in 50°C at pH 8.5 were purified using the HIC-HPLC system. When separated on native PAGE, only a single band of 95 kDa was visible (Srivastava et al 2009). Acting in parallel to the carboxy-terminal processing proteinase CtpA and the ATP-dependent thylakoid-bound metalloproteinase FtsH, the serine-type proteinase DegP is critical for the biogenesis and maintenance of PSII (Chi et al 2012).…”

Section: Resultsmentioning

confidence: 99%

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

2013

View full text Add to dashboard Cite

Proteolytic enzymes in wheat leaves were studied using zymographic detection of enzyme activities on one-way (1D) SDS-polyacrylamide gels and twodimensional (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 lM 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.

show abstract

“…The extract with Acetate-MS was particularly the buffer that showed the highest peak intensity ranging from 36 to 58 mAU, compared to the other extracts with Citrate-MS (28 to 37 mAU) and Tris-MS (12 to 19.5 mAU). Srivastava et al (2009) identified the presence of alkaline protease activity from spinach leaves by HPLC at 280 nm with less intense peaks in 2 to 4 min and predominantly in 7 to 9 min. It is important to note that the observed peaks can vary, as they are easily affected by experimental extraction parameters, type of sample, origin and plant conditions (Lombardo et al, 2010).…”

Section: High-performance Liquid Chromatography (Hplc)mentioning

confidence: 99%

Artichoke leaf extracts: Proteolytic activity, coagulant and HPLC analysis

et al. 2021

View full text Add to dashboard Cite

The search for origin plant-based proteases increases gradually due to their diversity and stability over a wide range of pH and temperature. Artichoke (Cynara scolymus) flowers are a proteolytic vegetable source already studied, but their leaves are scarce in this respect. Thus, the objective of this research was to obtain extracts of artichoke leaves with different buffers and extraction methods as an alternative proteolytic source and plant coagulant, as well as the separation and comparison of the protein profile of these extracts. The methodology used was based on extraction with sodium citrate buffer (pH 3), sodium acetate (pH 5) and Tris-HCl (pH 7) by mechanical stirrer (MS) and ultrasound (US); protein determination; proteolytic activity (PA) and specific activity (SA); milk clotting activity (MCA) and rennet substitution potential (RSP); high- performance liquid chromatography analysis (HPLC) with UV-Vis detector and principal component analysis (PCA). Extracts of Cynara scolymus leaves showed high results with Citrate-US for the parameters PA (14.38), SA (19.71), MCA (440) and RSP (30.60) compared to other treatments. The extracts with citrate and acetate presented a quick coagulation time (max 3 min). The HPLC analysis enabled the separation of the different protein compounds present in the extracts and most expressive peaks in the samples with Citrate-MS and Acetate-MS; and isolated peaks for Citrate-US. It was concluded that extracts of artichoke leaves with citrate and acetate buffer attributed satisfactory results to act as plant coagulant, as well as to carry out further studies for the purification of proteolytic enzymes and application in cheeses.

show abstract

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

Cited by 6 publications

References 33 publications

Plant senescence and proteolysis: two processes with one destiny

Plant senescence and proteolysis: two processes with one destiny

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

Artichoke leaf extracts: Proteolytic activity, coagulant and HPLC analysis

Contact Info

Product

Resources

About