Ashok, VASILI(MAJOR)Viroji Rao, S.T.Narasimha Reddy, YRAGA SUDHA, MEDICHERLA2018-10-052018-10-052007-09http://krishikosh.egranth.ac.in/handle/1/5810074849THESESABSTRACT: Presence of proteins in urine is considered pathological, but still certain proteins are excreted in minute quantities into the urine of healthy animals. The normal proteins in the urine are uroplakins (15 to 47kDa) and uromodulin (85 to 90kDa) which are produced by the urinary bladder and the thick ascending limb of Henle’s loop (kidney) respectively. The tissue specific expression of these proteins in the urinary system formed the basis for transforming the urinary system into a bioreactor to produce proteins of economic importance in the urine. In the present day world, the idea of transforming urinary system as a bioreactor and urine as an expression system has growing importance. So, the objective of the present study is isolation and quantification of these normal buffalo urinary proteins. The outline of the work is collection of urine from apparently healthy lactating Murrah buffaloes. Precipitation of proteins from the urine is carried out by protein precipitating agents like acetone, ammonium sulphate and sodium chloride. These precipitated proteins were analyzed by SDS-PAGE. Coomassie blue stained gel revealed a prominent and consistent protein band of molecular weight 85kDa in all the precipitated samples which could be uromodulin, the protein of our interest. Silver nitrate stained gels revealed low molecular weight protein bands (15, 27 and 47kDa) in all precipitated samples in addition to the high molecular weight band, which could be uroplakins. The consistent production of uromodulin by the animal into the urine gives an idea of transforming the kidney into a bioreactor. The reason for inconsistent occurrence of various low molecular weight proteins could be due to the dilution effect of liquid intake by the animal, diet, temperature, climate and various physiological factors which are yet to be ruled out. Quantification of protein precipitates of urine samples and urine as such of 10 different healthy buffaloes was done. The mean protein yields (mg) of urine and precipitates of acetone, ammonium sulphate and sodium chloride are 0.96±10.20, 10.60±0.39, 1.50±0.04 and 1.91±0.13 respectively. Acetone precipitation yielded significantly (p<0.001) higher quantity of protein precipitate. However, the banding pattern is not commensurate with the amount of precipitate. This could be the result of interference of salts and metabolic wastes with the Lowry’s method, yielding higher values. Ammonium sulphate precipitated more number of proteins, evidenced by the gel replicates, but at higher concentration. Sodium chloride selectively precipitates almost a single protein and is required in small quantities when compared to ammonium sulphate for producing good banding pattern. It is concluded that acetone precipitates higher amounts of protein quantitatively. Ammonium sulphate precipitates more number of proteins, where as sodium chloride precipitation yields almost a single protein. However, sodium chloride method has the advantage of selective precipitation over acetone and ammonium sulphateenThesis