Triglyceride lipase activities in rat liver

A kinetic study of neutral and alkaline triglyceride lipase activities from different liver homogenate fractions is reported. Lipase activities are studied with triolein as substrate and are determined by quantification of the released oleic acid liberated. Heparin-releasable, microsomal and mitochondrial lipase activities are studied as a function of time, protein concentration and substrate concentration. The neutral triglyceride lipase associated with mitochondrial membranes is kinetically different from the alkaline lipase, localized on the plasma membrane, which probably contaminates microsomal and soluble fractions.     

Effect of different collagenases on the isolation of viable hepatocytes from rat liver

The isolation of viable hepatocytes from rat liver was found to depend on the source of collagenase (EC 3.4.24.3) more than any other single factor examined. Collagenase purified from Achromobacter iophagus/Bacillus polymyxa (collagenase/dispase) gave reproducibly high viability without the use of complex perfusion protocols.     

Differentiation of microsomal from lysosomal triacylglycerol lipase activities in rat liver

The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.     

Fat-storing cells of the rat liver : Their isolation and purification

Fat-storing cells from the lobular area of the rat liver have been isolated by digesting the liver with pronase E and collagenase, and purified by Metrizamide density centrifugation and centrifugal elutriation. More than 70% of the cells in the final fraction were fat-storing cells. Per gram wet weight of liver, 3.1 ± 0.5 × 10⁶ cells were isolated. The purified cells showed a well preserved ultrastructure and contained lipid droplets with a fluorescence characteristic of vitamin A. A HPLC technique demonstrated the presence of large quantities of retinol and retinyl palmitate in the isolated fat-storing cells.     

Identification of a fibronectin receptor specific for rat liver endothelial cells

Antibodies raised against the fibronectin receptor of rat hepatocytes recognized one protein (Mr 120 and 135 kDa for unreduced and reduced samples, respectively) in immunoblotting of solubilized rat liver endothelial cells (LEC). The antibodies specifically precipitated a 200-kDa protein together with the 135-kDa component from 125I-labeled LEC. Spreading of LEC on fibronectin, but not on laminin or collagen, was inhibited by monovalent Fab fragments of the antibodies, implicating that the 135/200-kDa complex is a specific fibronectin receptor. The results indicate that LEC, hepatocytes, and fibroblasts of rat carry different fibronectin receptors, suggesting that the interaction of fibronectin with these cells may have different functional roles.     

Sialyl transferase activities of rat ascites hepatoma cells and rat liver

Sialyl transferase activities of the homogenate of rat ascites hepatome cells were compared with normal rat liver homogenate. The former had only 20% of the activity of the latter when an exogenous acceptor was added in the reaction mixture.Toward endogenous receptors, the activity of the hepatoma cell homogenate was 50% of that of the normal cell homogenate. A stimulation of the activity toward endogenous acceptors was observed when the homogenate of rat ascites hepatoma cells and that of rat liver were mixed. This stimulatory effect seems to be the consequence of utilization of acceptors from ascites hepatoma cells by the sialyl transferases of the rat liver.     

The isolation and characterization of specific 3-methylcholanthrene-binding proteins from rat liver cytosol

The major proteins to which 3-methylcholanthrene specifically binds have been purified over 480-fold with a 45% yield compared to a rat liver 100,000g supernate. The procedure involved a batch ion-exchange technique together with hydrophobic gel filtration and chromatofocusing chromatography. The multiple, specific 3-methylcholanthrene-binding proteins obtained from this protocol had apparent isoelectric points of pH 6.3, 6.0, 5.7, and 5.5 on elution from a chromatofocusing column. They all shared a common sedimentation coefficient as determined by sucrose gradient analysis of 4.4 S. Gel filtration on Sephadex G-75 gave a common Stokes radius of 27 A. An analysis of these chromatofocusing peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed those which eluted at pH 6.3 and 6.0 to contain two major protein bands of Mr 32,000 and 34,000, together with several contaminating proteins. In contrast, the peaks from chromatofocusing which eluted at pH 5.7 and 5.5 contained three major proteins of Mr 40,000, 25,000, and 14,000. The specific binding capability of these chromatofocusing peaks was found to be unstable to temperatures of -30 degrees C and below. Competition studies showed that these proteins were not steroid receptors, and that only polycyclic aromatic hydrocarbons which could induce cytochrome P-450c were able to displace 3-methylcholanthrene from the binding site. A marked preference was noted for polycyclic aromatic hydrocarbons with four to five benzene rings arranged in a nonlinear fashion, suggesting the stereochemical requirements of the protein binding site. The stability of the noncovalent interaction between the proteins and 3-methylcholanthrene was in the range of pH 7 to 9.     

A rapid method for the isolation of intact mitochondria from isolated rat liver cells.

A method is described for the preparation of intact mitochondria from isolated hepatocytesby sonication. Sonication of a suspension of rat liver cells for 10–30 s yields a homogenate from which tightly coupled mitochondria can be isolated. These mitochondria exhibit high respiratory control ratios and normal ADP:O ratios using glutamate plus malate, β-hydroxybutyrate, succinate, or ascorbate plus N,N,N′,N′-tetramethyl-p-phenylendiamine as substrates. The yield of mitochondrial protein is approximately 100–120 mg starting from 5 g of liver tissue. The mitochondrial fraction is essentially free of contaminating plasma membrane and microsomes and contains only small amounts of peroxisomes and lysosomes.