Activities of Proteolytic Lysosomal Enzymes in Blood and Liver of Growing Cattle

The activities of proteolytic lysosomal enzymes were investigated in serum, leucocytes, liver and muscle of German-Holstein heifers. Altogether there were 119 investigations: 19 9 months, 80 12 months and 20 16 months old female young cattle. Lysosomal enzymes in leucocytes were proven inappropriate for investigation because of very high variation coefficients. The enzymes investigated in serum, liver and muscle only had variation coefficients ranging from 20 to 40% in most cases. The closest connection between activities of individual enzymes was observed in the liver where activities, except for that of lysosomale esterase (EL), were highly correlated with each other. High correlations were calculated between 9 and 16 months old heifers.


Introduction
Differences between the protein yield in milk, or growth, can be observed between the periods of lactation or at a particular age and can be accounted for via the action of hormones (DAY et al., 1986;PANICKE, 1991;STAUFENBIEL, 1993;LACHMANN, 1994).The protein yield is the result of protein biosynthesis as well as proteolytic degradation.Degradation also determines the protein level in biological units.According to PFEIFER (1981), there is a close correlation between the activity of lysosomal enzymes and the concentration of protein in the cell.The proteolytic activity at a higher protein yield is restrained in all cells of the growing organism (SCHMIDT et al., 1992).Because this also holds true for leucocytes, the proteolytic activity could explain the different protein yields as well as the stability of the performance.The aim of investigation was to determine the correlation between the activities of individual lysosomal enzyme activities in serum, leucocytes, liver, and muscle as well as to correlate individual enzyme activities in the same compartment with different stages of cattle development.

Materials and methods
The proteolytic activities of lysosomal enzymes as well as their performance are influenced by systematic environmental factors.This is realized to a large extent by the simultaneousness and limited by the equipartition of the investigated young heifers of German-Holstein.Animals in groups 1, 3 and 4 were 10 to15 months old in different years.Heifers in group 2a were 9 months old , and those in group 2b were 16 months old.Groups 2a and 2b consisted of the same animals at different ages.Altogether there were 119 investigations in 99 female young cattle (Table 1).All animals lived in the same farm in different years.Samples of liver and muscle were obtained under sterile conditions by specific needle biopsy in conjunction with local cryoanesthesia (DIRKSEN, 1990).The tissue samples were washed free of blood with 0.9% NaCl at 4ºC.The tissue was transferred to a cooled homogeniser containing 3 ml 0.9% NaCl + 0.1% TRITON-X 100 and homogenised with a motor-driven Teflon-piston (800 rpm) at 4ºC for 3 minutes.The resulting homogenate was spun at 20,000 g for 20 minutes.The supernatant was stored at -20ºC for later enzymatic determination.The protein concentration of each supernatant was estimated by using the micro-Lowry-based Protein Assay provided by Sigma.Blood was taken from the vena jugularis of female young cattle and preserved with heparin.The isolation of the leucocytes from blood was performed using density gradient centrifugation (ZEMAN et al., 1988).A 1.5 ml volume of gradisol was added to a siliconized 12.5 ml centrifuge tube at 22 to 25 o C, followed by 2.5 ml of preserved blood.This was centrifuged at 400 g for 20 minutes in a swing-out centrifuge.The plasma phase was then poured off and the phase containing a mixed population of lymphocytes and granulocytes was removed using a Pasteur pipette.This was washed with 5 ml 0.9 % NaCl, suspended in 3 ml 0.1 % Triton X-100 and frozen.After thawing, the leucocytes were centrifuged at 20,000 g for 20 minutes and the clear phase was saved for enzymatic analysis.The activities of individual enzymes were determined using a sensitive spectrofluorimetric method using specific substrates (BARRETT, 1972).The activities of the enzymes in leucocytes, liver, and muscle were expressed as nmoles mg protein -1 h -1 , whereas the activity of cathepsin was expressed as mg degraded casein mg protein - 1 h -1 .In serum the activity was expressed as moles released l -1 h -1 .Investigated enzymes are listed below: The means and standard deviations were computed with the MEANS procedure, and the correlations with the CORR procedure of the SAS System, version 8.2 (SAS, 1999).

Results
The means and standard deviations of enzyme activities in serum, leucocyte, liver, and muscle were determined for each group investigated (Table 2).Leucocyte enzymes showed the largest standard deviations.No significant correlations were found between AGLD and other lysosomal enzymes in serum.LEU and SAAL activities were significantly correlated with only some enzymes in serum and leucocytes (Table 3).Medium correlation were found between daily gain and ALA and AGLD.In leukocytes ALA, LEU, and NAGL enzymes were correlated with each of the other lysosomal enzymes.The correlations were high in some cases.The enzyme most highly correlated with daily gain was ALA (Table 3).The statistical evaluation of enzyme activities in isolated tissues has limited implications as different reference systems are used.In the case of solid tissue or cell populations, the activity is expressed in terms of nmol or ng of degraded substrate per mg cytosolic protein.The reference system is the concentration of cytosolic protein, which can vary markedly between tissues and is affected by physiological conditions such as feeding, starvation, and diurnal fluctuations.These factors make precise evaluation of enzyme activities difficult.In serum, enzyme activity is expressed in nmol l -1 fluid -1 h -1 .Comparison of enzymatic activities between two different reference systems must be done with care.Correlations between the activities of eight lysosomal enzymes in liver and in muscle were estimated (Table 4).Both EL and daily gain were not significantly correlated with many of the enzymes.Other enzymes had correlation with each other enzyme in the liver and a negative correlation was found in case of EL with ARG and daily gain.Lysosomal enzymes were not significantly correlated with daily gain of the heifers in muscle.There were no high correlation found between enzymes in muscle except SAAL, which is the sum of ARG+ALA+LEU.
Table 2 Activity of lysosomal enzymes in serum (nmol l -1 h -1 ),leucocyte, liver, and muscle (nmol mg protein -1 h -1 )* (Aktivitäten der lysosomalen Enzyme in Serum (nmol l -1 h -1 ), Leukozyten, Leber und Muskel (nmol mg protein  The correlations between enzymes of different tissues are shown in Table 5 and 6.Where there were no correlations which differed significantly from zero, computed correlations were not reported in the tables.Activity of NAGL in serum resulted in negative correlation with enzymes in leucocyte.ARG, ALA and EL in serum had correlations with almost each enzyme in liver, but SAAL, NAGL and AGLD resulted in no correlations with liver enzymes.Lysosomal enzymes in muscle were not correlated with many of serum-and liver-enzymes, but most of its enzymes had correlations with ARG, LEU and EL in leucocyte.ARG and EL enzymes showed middle correlation with most lysosomal enzymes between serum-liver (negative correlations) and leucocyte-muscle.Some low correlations were determined between enzymes in leucocyte and in liver.

Discussion
The main site for the degradation of proteins is the lysosome compartment, which contains approximately 20 endo-and exopeptidases.Lysosomal proteolysis is strongly dependent on the type of cell and can account for 30 to 100% of the total proteolysis.Lysosomal degradation is somewhat specific, whereby proteins with longer half lives are preferred.Proteins with high turnover-rates are usually degraded by a ubiquitindependent mechanism in the proteosome.Various means of degradation are interconnected and strongly regulated.The assessment of proteolytic activity, particularly of lysosomal enzymes, provides insight on the level of protein synthesis.The factors affecting quality of cattle include growth, milk yield, fertility and health and are determined by genotype and environment.Consideration of proteinmetabolism is integral in assessing these parameters.Protein yield is the end result of proteinbiosynthesis and proteolysis.Proteolysis is restrained in all cells of developing organisms requiring higher protein yields.Due to external challenges, variances in proteolytic activities of leucocytes are especially high.There is high correlation between the quantity of protein in a given cell and the turnover rate.The rate of degradation of a protein appears to determine its final concentration.Protein degradation can occur in one of three ways: (BIENKOWSKI, 1983) via basal degradation in the endosomal system, intralysosomal proteolysis, or ubiquitindependent degradation in the cytoplasm (SOMMER and SEUFERT, 1992).It can be assumed that intracellular proteins are subject to constant turnover and that this process is physiologically important.In comparison to present knowledge concerning protein synthesis, the biochemical processes of protein degradation are relatively unknown (PANICKE et al., 1996).In terms of kinetics, the turnover of individual proteins is often described as the result of two zero order reactions for synthesis and one first order reaction for degradation (MILLWARD, 1978;AMENTA and BROCHER, 1981).This implies that the rate of protein degradation in a tissue or a population of cells is proportional to the concentration.Furthermore, it means that all protein molecules in the cell are equally prone to degradation.As a result, proteolysis occurs at the same rate in all cells at all times (AMENTA and BROCKER, 1981).The degradation of various protein molecules regulates important cellular functions such as structure, control of many key-enzymes and other bio-regulators, the elimination of abnormal protein molecules resulting from errors in synthesis, and the destruction of multi-complexes such as the ribosome or mitochondria.
To date, little research has been performed on the activities of lysosomal enzymes responsible for protein synthesis and energy production in dairy cattle.Because of the genetic determination of protein growth and energetic growth as well as the protein and energy turnover, genetic-physiological investigations seem to be confirmed by protein synthesis and proteolysis even concerning dairy cattle.Protein growth in different tissues results from a dynamic balance between the rates of protein synthesis and proteolysis (BIENKOWSKI, 1983;PANICKE et al., 1996).A shift in this balance towards restricted proteolysis results in increased protein concentration in the cells.This increase requires less energy in comparison to de novo protein biosynthesis, which requires relatively large quantities of ATP (BIENKOWSKI, 1983;BALLARD et al., 1980).Reduced proteolysis in cells, measured in terms of activities of the proteolytic enzymes, is an important genetic feature in selection and breeding (BALLARD et al., 1980;LOBLEY, 1998).Mammals reduce protein much more intracellularly than they absorb by feeding (LOBLEY, 1998).The lysosomal degradative enzymes are largely responsible for these processes, especially the proteases like Cathepsine D, L, B, H, and the amino-peptidases.Proteolytic activity in the cytosol is so low that the relatively high protein turnover of the cell cannot be explained by this alone.This proteolysis underlies the mechanism of cellular autophagy (SEGLEN and BOHLEY, 1992) and is under strict hormonal control.Thus, the activities of different proteolytic enzymes may vary during growth.The aim of this study was to gain further knowledge of the lysosomal enzyme activities in different tissues as serum, leucocytes, liver, and muscle.The proteolytic activities in these tissues were tested on growing HF-cattle (Holstein Friesian) at the age of 9 to 16 month in groups.
The following insights can be drawn from the results: 1.The phenotypic variation of the proteolytic activities of the enzymes within the groups, which are measured in serum as nmol l plasma -1 h -1 and in leucocytes, liver and muscle as nmol mg protein -1 h -1 , had a variation coefficient from 10 to 30 %. Similar variation has been observed for the milk yield with 22 up to 28 %. 2. The lysosomal enzyme activities of the leucocytes did not provide further insight for subsequent genetic investigations.They show a lower heritability coefficient (h 2 ), according to our own estimations on dairy cows and have much higher phenotypic variation (PANICKE et al., 1999).The latter point could be explained by the methodical limits in the separation of the leucocytes.3. The combination of the three aminopeptidase activities (ARG + ALA + LEU) as a common value (SAAL) seems to be justified as it decreases the variation coefficient to about 15%.The tight correlation coefficients between the individual values and their sum emphasize their equal importance in proteolysis as well as for their presentation in SAAL.In the serum, they reach about r=0.5-0.6, in the muscle and the leucocytes over r=0.8, and in the liver, over r=0.9.This fact may justify the application in breeding programs considerations.4. The tight relationship between the lysosomal enzyme activities in the liver exceeds that in the blood, leucocytes, and muscle.Even the CATH, which splits the molecules for the aminopeptidases, shows consistently tight correlation coefficients (over r=0.8).Between the ninth and the sixteenth month of age the correlations were tight (over r=0.8) for the nearly all of the enzyme activities.These correlations, in addition to the ensured relations to the serum, especially for ARG, ALA, and LEU, at about r=0.6, r=0.3, and r=0.3 respectively, emphasize a superior qualification of the liver parameters.We recommend the further investigation of the liver in physiological and genetic studies.The estimation of these parameters must be done under standardized environmental conditions and with suitable animals. 5.The derivation of suitable metabolic characteristics concerning the performance stability of domestic animals, including further direct or indirect liver parameters, requires more scientific investigations.