Seminal profiles of lysosomal enzymes in normal and infertile men.

Human seminal plasma is known to possess considerable proteolytic activity, much of which is associated with lysosomes. The activities of lysosomal hydrolases like alkaline proteinase, cathepsin-D, aryl-sulfatase and N-acetyl-beta-D-glucosaminidase in seminal plasma from randomly chosen infertile and vasectomised men have been compared. These enzymes have been implicated in the coagulation and liquefaction processes. The role of fructose and proteins in these processes has also been studied. The results indicate that cathepsin-D and aryl-sulfatase activity in infertile men were significantly lower than normo-spermic subjects. N-acetyl-beta-D-glucosaminidase was lowest in azoospermia suggesting that it could be used as a biochemical marker for azoospermia. Conversely, alkaline proteinase showed increased levels in all the infertile cases.

Purification and characterization of an alkaline proteinase from kidney cortex lysosomes.

An alkaline proteinase was purified to apparent homogeneity from buffalo (Bubalus bubalis) kidney cortex lysosomes by affinity chromatography on STI sepharose 4B and gel filtration over Sephadex G-100. The molecular weight of the enzyme was 17,000 and 21,000 by gel filtration and SDS/PAGE respectively. The purified enzyme was optimally active at pH 8.5-9.0 at 50 degrees C and hydrolysed synthetic substrates of chymotrypsin but not those of elastase or trypsin. It was inhibited by serine proteinase inhibitors like soybean trypsin inhibitor, limabean trypsin inhibitor and phenylmethyl sulphonyl fluoride. Immunologically, the enzyme was similar to chymotrypsin. The amino acid composition showed high content of acidic amino acids. This protein was detected in kidney, liver, spleen, pancreas and heart.

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation.

Highly pure lysosomes were isolated from buffalo (Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-ß-glucosamindase and cathepsin D in the lysosomal preparation were 26-45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3–1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure.