Fighting against POLLUTION to Save Environment

Factors affecting the proteolysis of cytoplasmic protein

Marath. Univ. J. of Sc. (Nat. Sci.) XVIII Sc.II, 25-28, 1979.
D. B. Boralkar and Usha R. Nagpal
Department of Botany, Marathwada University, Aurangabad.

ABSTRACT

Effects of cytokinin, gibberellic acid, vanillin, coumarin, 2-4 Dinitrophenol, Ascorbic acid, indole-3-acetic acid, pH and temperature on retention of cytoplasmic protein were investigated. It was observed that cytokinin, gibberellic acid, indole-3-acetic acid could prevent the breakdown of cytoplasmic protein exactly in the similar manner as in the leaf saps. Higher temperatures and acidic pH caused protein degradation, however, retention of cytoplasmic protein was noted at alkaline pH.

INTRODUCTION

Intensive work on agronomy of leaf protein has been done by earlier workers in this laboratory. This work led to the preparation and preservation of leaf protein concentrate for the ruminents and non-ruminents consumption. Preparation of leaf protein concentrate (LPC) involves many difficulties such as bringing vegetation to the laboratory and its complete processing. Preservation of leaf saps at different temperatures, pH and activators has been published (Usha Rani Batra, M. G. Deshmukh and R. N. Joshi, 1976).

The autolysis of protein in leaf extracts and proteolytic activity of crude preparations from such extracts against casein were studied by Singh (1962). He observed that on a hot day 40% of protein in young wheat leaves could be lost in 2 hrs. He used sodium thioglycolate and glutathione for avoiding the loss of protein. Cystine hydrochloride and potassium cyanide considerably increased hydrolysis. Osborne (1962) and Suiguira (1962) found that kinetin affected nucleic acid metabolism, causing an increase in RNA synthesis. Roger et al. (1969) found that sterile detached leaves when floated on water or kinetin changed total enzyme level. Cytokinins retard senescence in intact plants as well as detached leaves (Thimann et al., 1970). Activators and inhibitors used to prevent or promote protein breakdown have been studied by Thimann et al. (1970), Suiguria (1962), Singh (1962).

MATERIALS AND METHODS

Extraction of leaf saps was done with IBP pulper and press in laboratory conditions. The degradation in cytoplasmic fraction was studied in sannhemp and lucerne by heating juice at 50°–60° and removing chloroplastic protein fraction by centrifuging at 10,000 rpm. Supernatant contained cytoplasmic protein in soluble form and was used for the proteolytic studies. All this procedure took about 20 minutes.

2 ml of cytoplasmic fraction was taken in test tubes with 3 ml distilled water; suitable activators and inhibitors were added to give a final concentration of 0.002M. pH was adjusted with 1N HCl and 1N NaOH (pH 2, 4, 8 and 10). For temperature adjustment, cytoplasmic fractions were incubated at 40°, 60°, 80° and room temperature. In all experiments NPN (non-protein nitrogen) increase was determined by adding 10% TCA; protein nitrogen was determined by indirect calculations.

RESULTS AND DISCUSSION

In the above investigations maximum retention of cytoplasmic protein was noted with application of coumarin (62.8%) and then with kinetin (55.8%). Controls showed the highest degradation of about 50% in 3 hrs in sannhemp cytoplasmic fraction (Table I).

Increase of incubation time and temperature increased NPN and reduced protein level. At 80°, cytoplasmic fraction retained only 33.55% and 26.05% in sannhemp and lucerne respectively. Lowest degradation of protein was observed at room temperature i.e. 97% and 89.43% in lucerne and sannhemp (Tables II and IV).

Alkaline conditions retained protein to its maximum, but not acidic pH. Highest retention of cytoplasmic protein was observed at pH 10 in sannhemp and lucerne—92.24% and 66.19% respectively (Table III). Thimann et al. (1970, 1975) found 40% loss of cytoplasmic protein at pH 7.4 and 34% loss of chlorophyll at pH 5.

Table 1 : Effect of chemicals on cytoplasmic protein retention in Sannhemp

S.N.	Treatment	Initial NPN/ml	NPN/ml after 3 hrs	Initial PN/ml	PN/ml after 3 hrs	% Retention of PN
1	Control	0.60 mg	2.35	3.53	1.78	50.42
2	IAA		2.07		2.06	58.35
3	AA		2.04		2.09	59.20
4	Coumarin		1.91		2.22	62.88
5	2–4 DNP		2.08		2.05	58.07
6	GA		2.06		2.07	58.64
7	Kinetin		2.16		1.97	55.80