骨端軟骨部の基質小胞に関する酵素学的研究

Abstract

Matrix vesicles are membrane invested vesicles that initiate mineralization in the extracellular matrix of various calcifying tissues. Alkaline phosphatase (ALP) has been used as the marker enzyme of matrix vesicles, because matrix vesicles isolated from mineralizing tissues contain very high level of ALP in association with the plasma membrane of the cell. However, ALP is unsuitable as the marker enzyme of matrix vesicles because of its localization not only in the plasma membrane but also in other subcellular organelle membranes. Recently lactate dehydrogenase (LDH) isoenzymes were found to be located in matrix vesicles of the epiphyseal growth plate of young rabbit leg bones [Hosokawa et al. (1988) J. Biol. Chem. 263, 10045-10047]. In the present study, identification of lactate dehydrogenase isoenzymes in matrix vesicles was carried out with use of the epiphyseal cartilages from 6 different mammals. In some mammals, enzymatic properties of matrix vesicles in the growth cartilages were compared with those in the resting cartilages. 1. Matrix vesicles were isolated from the epiphyseal growth plates of 6 different young mammalian (dog, cat, rat, chicken, pig and monkey) rib bones. LDH activities were detected in the isolated matrix vesicles only in the presence of Triton X-100 but not in the absence of Triton X-100, showing that LDH is located in the matrix of the matrix vesicles. 2. The epiphyseal cartilages of young rat rib bones were divided into the growth zone and the resting zone, followed by the isolation and identification of matrix vesicles after collagenase treatment. Matrix vesicles with both ALP and LDH were also detected in the growth zone but were not detected in the resting zone. In contrast and surprisingly, LDH containing vesicles without ALP were found in the resting zone but not in the growth zone. The same results were obtained with the growth and resting cartilages of other young mammalian (dog, pig and monkey) rib bones, suggesting that LDH containing vesicles without ALP are also present in all mammals including humans. 3. Chondrocytes were prepared from the growth zone and the resting zone of the epiphyseal cartilages of young rat rib bones, homogenized with isotonic solution and subjected to sucrose density gradient centrifugation. In each case, LDH activity was recovered only in the soluble top fraction and not in sediment, showing that LDH is present only in the cytosol of chondrocytes but not in the plasma membrane and intracellular organelles. The results show that LDH in the vesicles is not from the plasma membranes and the membranes of intracellular organelles. 4. In both the growth and resting zone cartilages of young rat rib bones, isoenzyme patterns of LDH in the two different vesicles were identical with those of cytosolic LDH of chondrocytes. 5. Other cytosolic enzymes such as aldolase, aspartate : 2-oxoglutarate aminotransferase and alanine : 2-oxoglutarate aminotransferase were not detected in the two different vesicles from the growth and resting cartilages, suggesting the presence of a mechanism for the specific uptake of cytosolic LDH.