Studies on bovine and ovine rumen fl uid urease

Rumen fl uids were collected from a slaughterhouse. Mixed rumen contents of sheep and Holstein cows were transported to the laboratory in thermos fl asks, separately for each species. Ammonium sulphate and acetone were used for precipitation of protein in cell free rumen fl uid. The precipitate that was observed after freezing and thawing of cell free rumen fl uid and precipitate, obtained with 25% saturated ammonium sulphate showed no urease activity. Urease activity in precipitates from 80% saturated ammonium sulphate for cows and sheep were 3.6l and 6.17 (microgram ammoniaN, mg–1 protein. min–1), respectively. These amounts for acetone precipitate for cow and sheep were 1.88 and 0.48, respectively. Sodium dodecyl sulphate (SDS) acrylamide gel electrophoresis indicated the same subunit urease enzyme for sheep and cow. The molecular weight of rumen urease subunit was estimated to 75000 daltons, which is less than jack-bean urease (96000 daltons). Native urease did not separated by 3.2% acrylamide gel electrophoresis. The latter fi nding may indicate that rumen urease has a hexameric structure with more than 1500 kilodaltons.


INTRODUCTION
Urease is a nickel-containing enzyme that catalyses the hydrolysis of urea to form ammonia and carbamate.The latter compound spontaneously decomposes to generate a second molecule of ammonia and carbon dioxide.Bacterial urease plays an important role in ruminal nitrogen metabolism.Urea, supplied in the diet or recycled from saliva and the bloodstream, is hydrolysed to yield ammonia.The ammonia is then combined with ketoacids by the microbes to produce amino acids.Urease in the rumen will continue to convert urea to ammonia even if there is a lack of ketoacids.If the rate of ammonia production (i.e.urea via urease breakdown to ammonia) or ingestion of ammonia (i.e.ammoniated feeds) exceeds the microbes ability to utilize it to form amino acids, there is an accumulation of ruminal ammonia.The liver via the urea cycle normally detoxifi es ammonia absorbed from the rumen into systemic circulation.This detoxifi cation system can be overwhelmed resulting in elevated blood ammonia levels.In order to be able to control the activity of urease in the rumen, detailed knowledge about rumen urease is needed.In spite of the importance of the rumen urease in the physiology and nutrition, few studies are available.The presents report attempt to describe some of the properties of rumen urease.

MATERIAL AND METHODS
Rumen fl uids (one litre) were collected from an abattoir immediately after slaughtering animals.Mixed rumen content from native sheep and Holstein cows was squeezed through four layers of cheesecloth (separately for each species) and transported to the laboratory in thermos fl asks.All subsequent operations were conducted in cool condition (near to 4°C).The rumen fl uid was centrifuged at 6000 × g for 45 min and the clear supernatant was kept frozen overnight (-20°C).Next day, half part of the supernatant was fractionated with ammonium sulphate and another part precipitate with 30% acetone (separately for each species).The precipitates obtained with 25 to 80% ammonium sulphate and acetone were dissolved in sodium phosphate buffer pH=7 and subjected to heat treatment (20 min in 55°C).The mixture was centrifuged at 6000 × g for 50 min and supernatant was stored frozen at -80°C.

Acrylamide gel electrophoresis for urease
The supernatant was analysed at 4°C by acrylamide gel electrophoresis at pH 8.3 on 7.5 and 3.2% gel for native enzyme and electrophoresis was also done at pH 8.3 on 4% SDS-polyacrylamide gel.The SDS-polyacrylamide gel was stained with silver nitrate.

Assay of urease
Measurement of urease activity was done according to Moharrery and Das (2001).

Urease assay
After freezing and thawing in the fi rst step of sample preparation, a medium amount of precipitate was formed.This precipitate showed no urease activity.Precipitate, which was obtained with 25% ammonium sulphate showed very weak urease activity.In this regard, the precipitate formed after heat treatment showed no urease activity.The results from urease assay are presented in Table 1.This Table shows that the procedure for precipitation could be a factor, which BOVINE AND OVINE RUMEN FLUID UREASE affect urease activity in each species.In cows rumen fl uid the urease precipitate with acetone, showed higher urease activity than sheep rumen fl uid, but precipitate from ammonium sulphate in cows rumen fl uid, showed less urease activity compared to sheep rumen fl uid under the same condition.Generally the procedure with acetone precipitation has been shown not to be suitable for enzyme purifi cation and enzymes will lose their activity due to acetone action on enzyme protein denaturation, which cannot renaturate after disappearance of the acetone effect.3.60 a -µg ammonia-N min -1 b -µg ammonia-N min -1 • mg -1 protein c -urease precipitated with 30% acetone d -urease precipitated with 25 to 80% ammonium sulphate

Gel electrophoresis
The native urease enzyme from both species has not been separated by the acrylamide gel electrophoresis at pH 8.3 on 7.5 and 3.2% gel for native enzyme.The molecular weight for urease enzyme is near to 300 kDa, which can form a hexameric structure with more than 1500 kDa.In this condition separation of urease by gel electrophoresis, as explained earlier is not possible.When these type gels were stained catalytically for urease activity after electrophoresis, only band of urease activity appeared in the top of the gel, which indicated no separation has occurred during electrophoresis due to high molecule weight.These results are in contrast to the fi nding of Mahadevan et al. (1976) in which they separated bovine rumen enzymes by 7% acrylamide gel electrophoresis.
The subunit relative molecular mass for Jack-bean urease along with four types rumen urease were determined by using SDS-polyacrylamide gel electrophoresis.As shown in Figure 1, the rumen urease beside of species kind or procedure for precipitation, is homogeneous and has a subunit molecular weight of near to 75000 Da.The subunit molecular weight for rumen urease is less than Jack-bean urease (75 vs 96 kDa).The result of this report is agree with Hausinger (1986) who has reported 70000 ± 2000 daltons molecular weights for urease which is purifi ed from S. ruminantium.

Figure 1 .
Figure 1.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of urease.The gel was stained with silver nitrate and the dark area has been omitted from the fi gure for better view.Y axis shows molecular weight in kDa.Lines one to six are, standards, sheep, cow (ammonium sulphate), sheep, cow (acetone) and Jack-bean urease, respectively

Table 1 .
Urease activity in preparations from Jack-bean or rumen fl uid