Purification of beta-glucuronidase from urine of androgen-stimulated female mice

β-Glucuronidase (EC 3.2.1.31) has been purified from the urine of androgen-treated A/J female mice. This is a convenient starting material as the enzyme comprises 0.5 to 1.0% of total urinary proteins. Weekly injections of testosterone enanthate increased β-glucuronidase excretion from kidney into urine by approximately 300-fold. Unexpected urinary enzyme activity declined after six weekly treatments, but returned after the testosterone injections were discontinued. These observations suggest that testosterone influences not only the rate of β-glucuronidase synthesis but also the excretion of the enzyme into the urine. Other hormone regimens for achieving β-glucuronidase synthesis and excretion into urine are discussed. After concentrating the urine 10- to 12-fold, β-glucuronidase was isolated using two chromatography steps. Gel filtration on Bio-Gel A-0.5m resulted in a 16-fold purification of the enzyme and removed most of the major urinary proteins. Anion exchange on diethylaminoethyl-cellulose resulted in a further purification of β-glucuronidase by 12-fold. These two chromatography steps gave 190- to 200-fold increases of β-glucuronidase activity per milligram of protein and the enzyme electrophoresed as a single band in two native gels. However, analysis on sodium dodecyl sulfate gels revealed traces of protein smaller than the 70,000 molecular weight subunit of the enzyme. The β-glucuronidase isolated from urine had the same physical properties as the lysosomal form of the enzyme in mouse kidney.