Studies of the intercellular matrix of growth plates from dwarf and homozygous nonaffected alaskan malamutes: Collagen and hexosamine

Abstract

This study was performed to compare the extractability of dwarf growth plate collagen and hexosamine and that of homozygous nonaffected Malamutes and to measure the activity of three of the enzymes involved in the post-translational modifications of the collagen molecule. No significant differences were found in the activity of prolyl hydroxylase or lysyl oxidase in the dwarf growth plates. Lysyl hydroxylase activity in the dwarf was decreased to 22% and 33% that of the activity present in the homozygous nonaffected growth plates. Amino acid analysis of the collagen isolated from dwarf growth plates failed to reveal any decrease in hydroxylysine content. Growth plates were extracted with either 1 M sodium chloride or 4 M guanidine hydrochloride. The extracts were applied to a DEAE-cellulose column. Amino acid analyses of the material which did not bind to DEAE revealed a slight decrease in the amount of guanidine-extractable hydroxyproline in the dwarf but a 60-fold increase in the amount of salt-extractable hydroxyproline in the dwarf growth plates. Material which eluted with 1 M sodium choloride was analyzed for hexosamine. There was a 10-fold increase in the amount of salt-extractable hexosamine present in the dwarf growth plates, whereas no significant differences were observed in the guanidine-extracted material. Hexosamine analysis of the growth plates revealed a significant increase in the total amount of hexosamine present in the dwarf growth plates. SDS-polyacrylamide gels of the material which did not bind to DEAE as well as the pepsin digested, 0.9M sodium chloride precipitated collagen demonstrated the presence of only type II collagen.

Dwarfism in the Alaskan Malamute: Ultrastructural features of dwarf growth plate chondrocytes

“…These changes were related to the physiological increase in the level of protein synthesis within these cells. However, when marked and irregular dilations of the RER are observed in chondrocytes in growth plates from patients with endochondral growth defects, this finding is pathologic [8] and may reflect inability to transport material from the RER to the Golgi because of production of an abnormal product, a primary defect in the intracellular transport mechanism [18,34], or a rate of synthesis which exceeds the rate of intracellular transport from the cell. Dilated RER profiles have been found in chondrocytes from patients with several forms of spondyloepiphyseal dysplasia [22,26,29], Kniest syndrome [16,18,20], pseudoachondroplasia [11,20], metaphyseal dysostosis [9], thanatophoric dwarfism [21], and in the cho mutant in mice [27,28].…”

“…Solubility studies of homozygous nonaffected and dwarf Malamute growth plates have shown a significant increase in both total hexosamine and salt-extractable hexosamine in dwarf growth plates [8]. There is also an increase in the amount of salt-extractable collagen in the dwarf growth plates [8]. No differences were found in the activities of either prolyl hydroxylase or lysyl oxidase in dwarf growth plates.…”

“…A previous study utilizing routine transmission electron microscopy had failed to reveal any abnormalities in chondrocyte morphology [6]. Solubility studies of homozygous nonaffected and dwarf Malamute growth plates have shown a significant increase in both total hexosamine and salt-extractable hexosamine in dwarf growth plates [8]. There is also an increase in the amount of salt-extractable collagen in the dwarf growth plates [8].…”

“…No differences were found in the activities of either prolyl hydroxylase or lysyl oxidase in dwarf growth plates. Lysyl hydroxylase activity was decreased to 22% that of normal growth plates, but no decrease was found in the hydroxylysine content of whole dwarf plates or in the collagen isolated from dwarf growth plates [8].…”

Animal Models with Chondrodysplasia/Osteochondrodysplasm

“…Ultrastructurally, chondrocytes in the prolifer ative zone have dilated profiles of RER-containing granules presumed to be proteogly can granules, and clumps and nests of chon drocytes in the growth plate [8]. Investiga tions utilizing improved fixation methods (below) and extensions of biochemical stud ies of growth plate collagens [9] and proteo glycans [10] may identify the basic defect in this condition.…”