Purification of a low-molecular-weight phospholipase A(2) associated with soluble high-molecular-weight acidic proteins from rabbit nucleus pulposus and its comparison with a rabbit splenic group IIa phospholipase A(2).
An intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca(2+)-dependent phospholipase A(2) (PLA(2)) activity (about 70% of the total PLA(2) activity). The soluble PLA(2) activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA(2) was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW3000 column developed with a buffer containing 0.1-0.2 M salt. When the gel-filtration column was developed in the presence of 1 M salt, almost all the PLA(2) activity was eluted near the total available volume. The soluble PLA(2) was purified to near homogeneity. A Ca(2+)-dependent PLA(2) was also purified from the fractions extracted with 1 M KBr from nucleus pulposus. For comparison, we purified a Ca(2+)-dependent PLA(2) from the KBr fraction of spleen. The splenic PLA(2) was identical to a group IIa PLA(2), as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15. 7-kDa band at the same position as splenic group IIa PLA(2). These results suggest that group IIa PLA(2) is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.
Tanaka
N
N
Departments of Orthopaedic Surgery, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan.
Ishida
T
T
Hukuda
S
S
Horiike
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001965:Bromides / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002356:Cartilage / Q000033:anatomy & histology / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017680:Potassium Compounds / Q000737:chemistry; D011817:Rabbits; D013154:Spleen / Q000201:enzymology
D001965:Bromides; D046911:Macromolecular Substances; D017680:Potassium Compounds; D010741:Phospholipases A; C039004:potassium bromide; D002118:Calcium