K J Sparks

Immunochemical analysis of acetaminophen covalent binding to proteins: partial characterization of the major acetaminophen-binding liver proteins

A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.

Antibody binding of cartilage-specific proteoglycans.

Abstract Antiserum to cartilage-specific proteoglyean from chickens contains IgG which binds embryonic [ 35 S]sulfate-labeled cartilage-specific proteoglyean ([ 35 S]PGS I). The binding of antibody to [ 35 S]PGS I does not require prior digestion of the antigen with testicular hyaluronidase or chondroitinase ABC. Presence of [ 35 S]PGS I in the antigen-antibody reaction product was demonstrated using the precipitation method of Farr and confirmed by demonstrating that goat anti-rabbit IgG serum can precipitate rabbit anti PGS I—[ 35 S]PGS I complexes but not [ 35 S]PGS I alone. The antibody can bind essentially all (>90%) of the [ 35 S]PGS I. Antibody can bind to [ 35 S]PGS I in either the monomeric or the aggregated form. The antiserum does not contain antibodies to a number of glycosaminoglycans, to proteoglyean from skin or to a minor proteoglyean component of cartilage (PGS II). The antiserum cross-reacts with cartilage-specific proteoglycan from a different avian genus, the Japanese quail.

Preparation and characterization of an antiserum against purified proteoglycan link proteins from avian cartilage.

Link proteins have been purified from avian xyphoid process. Cartilage was extracted in 4.0 M guanidine hydrochloride and a link fraction (A1D5) was obtained by sequential cesium chloride centrifugation. Link proteins were separated from low buoyant density proteoglycans by chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The presence of contaminating proteoglycans at various purification steps was monitored in an enzyme-linked immunosorbent assay using antiserum against avian cartilage proteoglycan monomer (anti A1D1-1400 Vo). Antiserum generated against this purified link preparation (anti link[SDS]) was characterized for its ability to bind link proteins, proteoglycan monomer, and aggregate. The serum was specific only for link proteins when tested by an enzyme-linked immunosorbent assay. Some reactivity against proteoglycan monomer was observed in a Farr-type assay.

Physical and immunochemical properties of low-buoyant-density proteoglycans from avian cartilage

Abstract A proteoglycan link fraction (A 1 D 5 ) from avian xyphoid cartilage contains one link protein and low-buoyant-density proteoglycans. An antiserum made against this fraction (anti-A 1 D 5 serum) specifically binds [ 35 S]sulfate-labeled proteoglycans from aggregate (A 1 ), monomer (A 1 D 1 ), and link (A 1 D 5 ) fractions synthesized by 14-day embryonic chick sterna. When [ 35 S]sulfate-labeled proteoglycans from either monomer (A 1 D 1 ) or link (A 1 D 5 ) fractions are used as the antigen, anti-A 1 D 5 serum binds as well as an antiserum against a purified monomer preparation (anti-A 1 D 1 1400 Vo serum). However, reduction and alkylation of the proteoglycans from the monomer fraction does reveal differences in the binding of the two antisera. Whereas reduction and alkylation of the antigen ( 35 S-A 1 D 1 ) inhibit binding by both antisera, binding of the anti-A 1 D 5 serum is more affected by antigen alteration. Differences in the two antisera can also be detected in assays using labeled aggregate as antigen. Anti-A 1 D 5 serum cannot maintain the same levels of antigen binding as the anti-A 1 D 1 -1400 Vo serum when compared in an antibody dilution curve. In addition, anti-A 1 D 1 -1400 Vo serum can stabilize aggregate during sedimentation in a sucrose density gradient containing 4 m guanidine hydrochloride whereas anti-A 1 D 5 serum cannot. These results suggest that the low-buoyant-density proteoglycans found in the link (A 1 D 5 ) fraction from avian cartilage contain some of the antigenic determinants found on the proteoglycan monomer. However, there is at least one monomer determinant which is not present in the A 1 D 5 fraction.