Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Demonstration of a rat liver microsomal binding protein specific for beta-glucuronidase.

A binding protein with apparent specificity for beta-glucuronidase has been partially purified from a Triton X-100 extract of rat liver microsomes by affinity chromatography on glucuronidase-Sepharose 2B. It appears that once removed from the membrane, this binding protein self-aggregates to form large macromolecular complexes. With the use of polyacrylamide gel electrophoretic and sucrose density gradient ultracentrifugation assays to monitor the conversion of glucuronidase tetramer to a very high molecular weight complex, it was shown that the binding activity is heatlabile and protease-sensitive. However, binding activity is not influenced by salts, carbohydrates, other proteins or glycoproteins, or by extensive periodate oxidation of beta-glucuronidase, nor does binding occur with any other protein tested. The binding protein does not discriminate against any form of beta-glucuronidase from any rat organ tested. However, the binding protein does show organ localization, being present in the liver and kidney but not the spleen. The possible relationship of this binding protein to egasyn, a membrane protein which stabilizes beta-glucuronidase in mouse liver endoplasmic reticulum, is discussed.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases

The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)_0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)₄[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver.     

Characterization of the binding of human growth hormone to microsomal membranes from rat liver.

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 X 10(10) +/- 0.2 X 10(10)M-1 and 0.03 X 10(10) +/- 0.007 X 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.     

Net glucuronidation in different rat strains: importance of microsomal beta-glucuronidase

The net glucuronidation of bilirubin (BR) has been determined in inbred and outbred rat strains and their subpopulations with similar glucuronosyltransferase (EC 2.4.1.17) activity but with different levels of beta-glucuronidase (beta G) (EC 3.2.1.31), or in which the level of beta G activity was reduced with D-glucaro-1,4-lactone. These studies demonstrated that outbred rat strains consist of two subpopulations that differ approximately 1.5- to two-fold in serum and liver beta G activity. Evidence is presented indicating that owing to its compartmentalization the lysosomal beta G, unlike the corresponding microsomal enzyme, is neither inhibited by glucarolactone nor accessible for hydrolysis of newly synthesized glucuronides. The ratio of glucuronidated to unconjugated BR 15 min after injection of albumin-bound BR into the tail vein appears to correlate negatively with the liver microsomal beta G activity. The results may be relevant to the relative risk to toxins, including carcinogens, and to their reduction by dietary intervention.     

Characterization of microsomal NADPH-dependent aldehyde reductase from rat brain

An NADPH-dependent aldehyde reductase was purified from rat brain microsomes to electrophoretic homogeneity. The purified enzyme had a molecular weight of 75,000 and reduced long chain fatty aldehydes such as octanal and hexadecanal with higher affinity (Km values of 0.21 mM and 0.03 mM, respectively) than for various artificial carbonyl compounds such as p-nitrobenzaldehyde and p-nitroacetophenone (Km values of 0.31 mM and 1.4 mM, respectively). The purified microsomal aldehyde reductase also showed NADPH-cytochrome c reductase activity, and it could not be distinguished from NADPH-cytochrome c reductase in molecular weight (75,000), chromatographic behavior, electrophoretic mobility, or immunological properties. The solubilized microsomal fraction treated with steapsin lost the reductase activity for hexadecanal but not that for cytochrome c. These results suggest that the aldehyde reductase in brain microsomes is identical to NADPH-cytochrome c reductase and that a hydrophobic portion of the NADPH-cytochrome c reductase is required for the reduction of hexadecanal.     

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Comparison of nuclear and microsomal epoxide hydrase from rat liver

The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30–60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (? 49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.