Investigation on proteolitik enzyme activity and protein mobilization of Vicia faba L. seeds


Thesis Type: Doctorate

Institution Of The Thesis: Uludağ Üniversitesi, Turkey

Approval Date: 2003

Thesis Language: Turkish

Student: SERAP KIRMIZI

Supervisor: GÜRCAN GÜLERYÜZ

Abstract:

Tn this study it was aimed to show changes in protein mobilization and proteolytic activity of the Vicia faba L. cv. Eresen 87 (Fabaceae; Leguminosae) secds which is known to have high protein content. The working procedure was planned to determine the effect of the embryonic axis and the effects of plant grovvth regulators (GA3, BA, IAA and ABA) on Endopeptidase (EP), Leucine aminopeptidse (LAP), Carboxypeptidse (CP) activities, changes in total soluble proteins and free amino acid content during the 7 days period. As well as the SDS-PAGE was used to examine the the changes in total soluble proteins during the germination period. The seeds were sovvn as attached (normal) and detached cotyledons in petri dishes and incubated with vvater or plant grovvth regulators at 23 °C darkness, during the 0,1,3,5,7 days period. Detached cotyledons vvere incubated for 3 and 5 days with plant grovvth regulators to determine vvhether the plant grovvth regulators can take place of the embryonic axis. Asocasein for EP, L-Leucine 4-nitroanilide for LAP, and N-CBZ Phe-ala vvas used for CP activity as substratum. The crude homogenate vvas used as source of enzyme, and it vvas incubated for a certain period of time in a vvater bath. After termination of reaction, the activity vvas determined with spectrophotometric method. EP activity vvas defined as Units/hour, LAP activity vvas defined as released p- Nitroanilide/cotyledon, CP activity vvas defined as released mM Leucine equivalents/cotyledon. Total soluble proteins determined with Coomasie Brilliant Blue stain with BSA(Bovine Serum Albumine) standart and expressed as mg/cotyledon. The ninhydrine reaction vvas done for determining the free amino acid contents of the homogenate and it vvas expresscd as mM Leucine/cotyledon. Total soluble protein content has decreased in both attached and detached cotyledons as the germination proceeds. Free amino acid content has increased in attached cotyledons but not detached cotyledons as the germination proceeds. The negative significant correlation vvas found betwecn total soluble protein and free amino acid content in attached cotyledons. It vvas concluded that the protein mobilization not completely supressed vvhen the embryonic axis detached from the cotyledons. Among the three proteolytic enzymes, only EP activity vvas found to be affected by removal of the embryıonic axis. The EP activity vvas lovv at the beginning of germination but increased during the incubation period in both attached and detached cotyledons. LAP activity was low at the beginning of the germination but decreased as the germination proceeds both in attached and detached cotyledons. CP activity was reached maximum at the day 5 th, then decreased in both attached and detached cotyledons. Among the used plant growth regulators (104 M GA3, BA, IAA and 10'5M ABA), only BA was found to cause an increase of the LAP activity and total soluble protein content.According to results from SDS-PAGE, protein mobilization was started at the day 3rd in attached cotyledons and the 7th day for detached ones. The positive effects of plant growth regulators has been seen, hovevver it was concluded from the knowledge of the literatüre the effects of the plant grovvth regulators could be specific to species and also cultural variety.While dry weight of the attached cotyledons decreased, the dry weight of the embryonic axis increased as the germination proceeds. Probably, this could be due to mobilization of the storage reserves from cotyledons to embryonic axis.