Influence of protein starvation on some lysosomal enzyme activities in blood serum of sheep

Titel der Arbeit: Der Einfluss von Eiweißmangel auf die Aktivität von lysosomalen Enzymen im Blutspiegel beim Schaf Die Aktivitäten der lysomalen Enzyme wurden in Blutplasma der Schafe nach 48 stündiger Eiweißhungerperiode bestimmt. Die Aktivitäten von BGLU, BGAL, AAP und LAP erhöhten sich, die von NAGL und LL verminderten sich signifikant. Keine Veränderungen zeigten die Aktivitäten von EL und KF.


Introduction
The role of the cell lysosomal compartment in the processes of protein degradation is connected with the adaptation ability of an organism (BAIRATI et al., 1997;MIYAWAKI, 1988;PFEFFER, 1991;SEGLEN and BOHLEY, 1992;STORRIE, 1995;STORRIE and DESJARDINS, 1996).Lysosomal enzymes may constitute a good model for studies of these adaptation processes on the cell level (BRUNK et al., 1996;CALDER, 1989;DESJARDINS, 1995;HICKS, 1995;KOŁĄTAJ et al., 1996;LOMBARDO et al., 1996;WITEK et al., 1994;1995;1996).Also the problem of stress in animal breeding is becoming an important research problem, connected with its negative effects on productivity (HERSHOCK and  VOGEL, 1989; KOŁĄTAJ et al., 1998; 1996; 1995; ).In the literature available no information was found on lysosomal enzyme activities in the tissues of sheep, which would present the animals in the physiological conditions and in response to stress.WITEK et al. (1996) observed the activity of lysosome enzymes in leucocytes and blood plasma of sheep as result of mercury poisoning only.The present studies are connected with the activity of model lysosomal enzymes in the blood plasma of sheep maintained in farm conditions, fed normally and next restrictived for a short period.Fasting or restriction of food is some specific type of stress (FIDANZA, 1980;KOŁĄTAJ, 1993).In domestic animals it still occurs quite often (KLUSEK et al., 1997).

Material and Methods
The studies were performed on 21 Booroola rams 1-4 year old (Table 1) which came from the breeding farm belonging to the Polish Academy of Sciences, Institute of Genetics and Animal Breeding in Jastrzębiec.The animals received unlimited hay, silage and concentrate according to norm for farm.They had a constant access to water.All the animals were in good conditions of breeding, nursing and professional veterinary care.x \ significance of differences between means was estimated according to the contrast method; The animals maintained in these conditions were treated as blood donors of the control group.Next, the animals were deprived for 48 hours the silage and concentrate, therefore the protein components but they had a constant access to straw only.We did not apply the full starvation in order to not evoke the emotional stress connected with a total lack of food.The essence of our intention was a deprivation of food protein during this period.After 48 hours of that protein starvation the blood was drawn again.Both times, before 48 h period of food restriction (control) and after this time, 10 ml of blood were drawn between 8 30 -10 30 a.m. to test tubes containing heparin and centrifuged for 15 minutes at 20.000 g in K-24 centrifuge.The blood samples were taken from the neck vein of all individuals.In the blood serum obtained the following lysosomal enzymes were determined: ß-glucoronidase (BGRD) -EC 3.  method (1960).The enzymatic activity was expressed in nMol/mg of protein/hour.Plasma protein was estimated according to the method of KIRSCHKE and WIEDERANDERS (1984).
The reagents used were produced by Sigma (Sigma -Aldrich Corp.) firm.The results obtained were analysed statistically using the two-way analysis of variance.

Results
The results obtained are presented in Tables 1 -3 and Figure 1.After 48 hours of protein fasting increased significantly the activity of BGLU (F=12.385),BGAL (F=4.280),AAP (F=20.516),LAP (F=46.216).The activity of NAGL (F= 55.356) and LL (F=6.675)decreased significantly while BGRD, EL and KF remained unchanged.The analysis of variance (Table 3) indicated, that as regards NAGL and LL the age of the animals played a significant role in determining the enzyme activity (F=10.984,P ≤ 0.001 and F=3.302, P ≤ 0.03, respectively).The age x treatment interaction revealed the significant value (F=3.244,P<0.034) only in relation to acid phoshatase -KF (Fig. ).

Discussion
Stress has already been defined many times (FRIEND, 1980;MOBERG, 1985;SPENCER, 1995;YOUSEF, 1985).There are the suggestions that stress should be understood as the sudden informatic and energetic excitation of the cell or the organism as a whole (KOŁĄTAJ, 1993).It is known that numerous regulatory possibilities of homeostasis reveal under the pressure of environment (KLASSING, 1985).An analysis of our results obtained indicates that 48 h protein fasting is an important stress factor in relation to lysosomal enzymes and it can cause the changes of their activities.The activity of four enzymes investigated increased and the activity of two enzymes decreased.The activity of BGLU, BGAL, AAP and LAP enzymes increased.It may be connected probably with the degradation intensity of protein and protein -carbohydrate compounds in the cell.Such selective changes may suggest the different of breaking stages of the lysosome arrangement membranes.This phenomenon may be treated as an example of the adaptation of the cell lysosome arrangement to conditions of 48 h lasted protein fasting.In this period the glycoprotein and lipoprotein metabolism rate increases, as well as the rate of passage of aminoacids from the degraded proteins to mitochondria in order to maintain a correct level of oxidative phosphorylation.It is known that different types of proteins have a different period of half-life.It proves to their differently controlled degradation because metabolism must take place also during protein fasting.The problems of human and animal adaptation to the starvation and to the restricted feeding remain in the centre of interest of many researches (CHEREL et al., 1992;FIDANZA, 1980;KOŁĄTAJ, 1993;WITEK et al., 1995).

Figure :
Figure:The example of interaction treatment x age for acid phosphatase (KF) activity

Table 2
The example of the interaction (in one case only) for least squares means (LSM) and (± se) for activity of studied lysosomal enzyme KF x\ x \ significance of differences between means was estimated by Duncan's test;