Ovine Bone Marrow Extracellular Matrix and Soluble Protein Extraction: Fetuin Amino Terminus Microheterogeneity

Abstract

In this article, the authors describe a new method using phosphate-buffered saline for the initial extraction of extracellular matrix (ECM) and soluble proteins from hematologic tissues. Direct comparisons between this method and previously reported methods demonstrate superior total protein yields with the novel technique in a fraction of the time for these ovine hematologic tissues: bone marrow, marrow aspirate, spleen, liver, and blood. Analysis by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining demonstrate comparable protein patterns with this method and a previously reported method. This method also successfully extracts the ECM glycoproteins fibronectin and laminin as well as the proteoglycan, chondroitin sulfate from marrow. These findings are demonstrated by Western and dot blotting. Bone marrow ECM and soluble proteins extracted by the novel method stimulate myeloid progenitor growth in a methylcellulose assay. Using an assay for elastase inhibitory capacity, the authors demonstrate that alpha 1-proteinase inhibitor, the principal inhibitor of neutrophil elastase, is present and that its activity is preserved in bone marrow samples extracted with this team's method. In contrast, very low total protein yields are obtained with the method used previously to recover hemonectin, a rabbit bone marrow ECM granulocytic cytoadhesion molecule. This team's novel procedure, which extracts ECM and soluble proteins from small samples of tissue in a rapid, efficient, and reproducible manner, greatly enhances the analysis of these proteins from tissue culture, animal, and human clinical samples. In addition, with purification by chromatofocusing chromatography and molecular sieving gel electrophoresis, N-terminal amino acid sequencing could be performed on a developmentally regulated marrow protein with biochemical properties similar to those of hemonectin and the plasma protein fetuin. The authors propose that this novel technique be used for the initial extraction of ECM and soluble proteins from hematologic tissues and that subsequent, definitive recovery of insoluble proteins be accomplished using previously reported, though less efficient, methods.