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, Marathdwada University, Aurangabad.

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

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) invovles 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 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 kinetinaffected nucleic acid metabolism, caused an increase in the synthesis of RNA. 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 inhibitotrs.used to prevent or promote protein breakdown. It hasbeen proved by Thimann et al. (1970), Suiguria (1962), Singh (1962).

Extraction of leaf saps was done with IBP pulper and press in the laboratory conditions. The degradation in cytoplasmic fraction was studied in sannhemp and lucerne by heating juice at 500-600 and removing chloroplastic protein fraction by centrifuging at, 10,000 rpm. Supernatant contained cytoplasmic protein in a soluble from and was used for the proteolytic studies. All this procedure took about 20 minutes. 2ml of cytoplasmic fraction was taken in test tubes with 3 ml distilled water, sutitable activators and inhibitors were added to give a final concentration of 0.002M. pH was adjusted with 1N HC1 and 1N NaoH 2, 4, 8 and 10 for temperature adjustment cytoplasmic fractins were incubated in the incubators at 40°, 60°,80° and room temperature. In all the experiments NPN (non-protein nitrogen) increase was determined by adding 10% TCA, protein nitrogen was determined by inditrect calculations.

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 degration of about 50% in 3 hrs in sannhemp cytoplasmic Fraction ( Table I).

Increase of incubation time and temperature increased NPN and brought down the 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 tempreature i. e. 97% and 89.43% in lucerne and sannhemp (Table II and IV ) .

Alkaline conditions could retain protein to its maximum, but not the acidic pH Highest retention of cytoplasmic protein was observed at pH 10 in sannhemp and lucerne,and lucer 92.24% and 66.19% respectively (Table III). Similar studies were carried on by Thimannet al. (1970, 1975) on oat leaves. They found 40% loss of cytoplasmic protein at pH 7.4 and 34s% loss of chlorophyll at pH 5.

Table 1 : Effect of chemicals on cytoplasmic protien retention in Sannhep.

S. N. Treatment  Initial
NPN /ml
NPN /ml
after 3 hrs
 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

IAA - Indole - 3 - acetic acid       2-4 DNA - 2-4 Dinitrophenol
AA - Ascorbic acid.                     GA3 - Gibberellic acid.