Rabbit knee articular cartilage was studied after the joint was submitted to immobilization, running or increased weight bearing for 24-27 days. Immobilization with a plastic splint reduced the fraction of proteoglycans not extractable with 4 M guanidinium chloride (GdnHCl). In the immobilized joints the chondroitin sulfate content was elevated as calculated from the galactosamine/glucosamine ratio. The ability of these proteoglycans to reform aggregates with endogenous hyaluronic acid in Sepharose CL-2B chromatography was reduced. Increased exercise was associated with an elevation of proteoglycans extractable with 4 M GdnHCl. The increased weight bearing occurring in the contralateral knee elevated the content of proteoglycans not extractable with 4 M GdnHCl. Other effects of weight bearing included increased glucosamine concentration, suggesting accumulation of keratan sulfate-rich proteoglycans, and an elevated hydroxyproline concentration.
A synthetic substrate (p-nitrophenyl-\g=a\-D-glucopyranoside) was used to measure the acid and neutral \g=a\-glucosidaseactivity in bull seminal plasma, spermatozoa and in homogenates of bull reproductive organs. Marked differences were observed in the activities of these enzymes in the various tissues studied. Epididymis and particularly its caput region contained the highest specific activity of acid \g=a\-glucosidase. The activity of neutral \g=a\-glucosidase was highest in testis and in different parts of the epididymis. Seminal plasma, spermatozoa and seminal vesicle secretion contained only the acid enzyme activity.After fractionation with anion exchange chromatography in HPLC (Mono Q) and chromatofocussing, acid \g=a\-glucosidaseactivity of seminal plasma was recovered in two fractions with different pI values. The corresponding activities were found in the secretion of seminal vesicles, which thus form the major secretory source of seminal plasma acid \g=a\-glucosidase. In the fractionation with gel filtration on Sepharose 6B, the acid \g=a\-glucosidase had a smaller molecular weight than did the neutral enzyme. In anion exchange chromatography and chromatofocussing the testicular and epididymal homogenates each contained two acid and two neutral isoenzymes. In both fractionations the elution pattern of acid \g=a\-glucosidase was clearly different from that of the enzymes in seminal plasma.The pH optimum of acid \g=a\-glucosidaseranged from 3\m=.\75to 4\m=.\5and that of the neutral enzyme from 6\m=.\5 to 7\m=.\0. The neutral activity was more sensitive to many divalent metal ions and differences were also observed in the response of the enzymes to different concentrations of turanose and KCl.
The highest specific activity of beta-N-acetylglucosaminidase (beta-NAG) was found in the different parts of the epididymis, where the activity seemed to be partly in secretory and partly in non-secretory, tissue-bound form. Epididymal spermatozoa also contained moderate beta-NAG activity. The beta-NAG was separated by chromatofocussing and anion exchange chromatography with HPLC into multiple forms with distinct pI values (8.0-4.0). The cauda epididymidis, ampulla and the seminal vesicles formed the major secretory sources of the high beta-NAG activity in bull seminal plasma. The major secretory forms of beta-NAG in caput and cauda epididymidis showed distinct elution profiles. In the fractionation with gel filtration on Sepharose 6B, the beta-NAG activities derived from bull testis and caput epididymidis had smaller molecular weights than did the secretory enzymes in seminal plasma, seminal vesicle secretion and cauda epididymidis. Maximum activity of all beta-NAG isoenzymes was observed at pH 5.0. They were almost totally inactivated at 60 degrees C and about 75-80% of the activity was lost at 55 degrees C. All the isoenzymes were strongly inhibited by thiol reagents but not with other metal ions and chelating agents. Histochemical studies showed a strong granular (lysosomal) reaction for beta-NAG in basal cells and basal parts of the principal cells in all but the initial segment of the epididymis. An apical (secretory) reaction was prominent in the distal caput and corpus as well as in distal cauda. After the distal caput the luminal sperm mass became increasingly mixed with a beta-NAG-positive material. The epithelial cells of the ampulla and seminal vesicle displayed a moderate apical (secretory) reaction.
Acid phosphatases of the rat ventral prostate were fractionated by gel filtration (GF) on Sepharose 6B, isoelectric focusing (IEF), and chromatofocusing (CF). In GF three activity peaks (GF-1, GF-2, GF-3) were disclosed. They showed some differences in substrate preference when six substrates (p-nitrophenyl phosphate; p-NPP; phenolphthalein phosphate, Phe-P; thymolphthalein phosphate, Tym-P; alpha-naphthyl phosphate, alpha-NP; beta-naphthyl phosphate, beta-NP; naphthol ASBI phosphate, N-ASBI-P) were tested. Differences were also encountered in their sensitivity to tartrate and fluoride. IEF gave seven bands at different pI values (8.3, 8.1, 7.9, 7.1, 6.4, 5.5, and 5.0) with alpha-NP and beta-NP but only four with N-ASBI-P. Four of the bands (8.3, 8.1, 7.9, 5.5) were sensitive to tartrate. In CF eight activity peaks (CF-1 to CF-8) were resolved with the six substrates. They differed from each other in pI values, pH optima, substrate preference, and modifier characteristics. Peaks CF-1 (pI 8.3, pH 5.5), CF-2 (pI 8.1, pH 4.2) and CF-3 (pI 7.9, pH 4.2) had a large substrate spectrum and high sensitivity to tartrate and fluoride. CF-4 (pI 7.1, pH 6.0) and CF-7 (pI 5.5, pH 4.2) were low in activity, preferred alpha-NP as substrate, and were moderately sensitive to tartrate. CF-5 (pI 6.4, pH 5.5) and CF-8 (pI 5.0, pH 5.0) were able to hydrolyse all substrates tested with moderate inhibition by tartrate. CF-6 (pI 6.0, pH 5.0) showed a relative preference for p-NPP and Phe-P with no hydrolysis of N-ASBI-P and Tym-P. Of these activities CF-6 and CF-7 were also clearly activated by Co2+. Peaks CF-6 and CF-7 appeared the most sensitive to p-chloromercuribenzoate. It is concluded that activities CF-1, CF-2, and CF-3 are lysosomal isoenzymes with minor structural differences. The others are possibly all nonlysosomal with greater biochemical differences. Some of them apparently represent the secretory form(s) of acid phosphatase in the rat ventral prostate.
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