Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269–2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 μm to 0.11 μm in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A₂ activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A₂ is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.     

Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.     

Isolation and characterization of a NADH-dehydrogenase from rat liver mitochondria

Mitochondrial NADH dehydrogenase has been purified from rat liver mitochondria by protamine sulfate fractionation and DEAE-Sephadex chromatography. The enzyme is water-soluble and its molecular weight has been estimated at 400 +/- 50 kilodaltons. NADH-ferricyanide reductase and NADH cytochrome c reductase activities have been studied and the kinetic parameters have been determined. Both substrates, NADH and the electron acceptor (ferricyanide or cytochrome c) have an inhibitor effect on the reductase activities and the kinetic mechanism of the enzyme is ping-pong bi-bi.     

Isolation and characterization of mucopolysaccharides from rat liver mitochondria.

Mucopolysaccharides were isolated from rat liver mitochondria which had been labeled with 35S-sulfate. They were prepared from trichloroacetic acid (TCA)-insoluble and -soluble fractions of lipid-free mitochondria. These fractions were digested with pronase exhaustively, and the mucopolysaccharides were recovered in the void volume fractions of gel filtration of the pronase digests on Sephadex G-50, monitored by radioactivity determination. Identification of these mucopolysaccharides was based on electrophoresis on cellulose acetate film using three different media, enzymatic and chemical degradations specific to each type of mucopolysaccharide, using chondroitinases, heparitinase, and nitrous acid. From the TCA-insoluble fraction, chondroitin sulfate A and dermatan sulfate were obtained in a ratio of about 1 : 2, based on 35S-radioactivities, whereas the TCA-soluble fraction yielded chondroitin sulfates A/C, dermatan sulfate, and heparan sulfate in a ratio of about 1 : 3 : 12. The total amount of mitochondrial mucopolysaccharides was about 3 mg/g protein, distributed between the TCA-insoluble and -soluble fractions in a ratio of about 1 : 3.     

Isolation and characterization of DL-methylmalonyl-coenzyme A racemase from rat liver

Certain amino acids and other compounds are metabolized via propionyl-CoA----D-methylmalonyl-CoA----L-methylmalonyl- CoA----succinyl-CoA----tricarboxylic acid cycle. D-Methylmalonyl-CoA can also be converted to methylmalonic acid and coenzyme A by a specific hydrolase that does not act on L-methylmalonyl-CoA [R.J. Kovachy, S.D. Copley, and R.H. Allen (1983) J. Biol. Chem. 258, 11415-11421]. Because little is known about mammalian DL-methylmalonyl-CoA racemase and because it is involved in the flow of D-methylmalonyl-CoA to L-methylmalonyl-CoA----tricarboxylic acid cycle (versus to methylmalonic acid), we developed a new assay and purified rat liver racemase 23,000-fold to homogeneity. The molecular weight of the racemase is 32,000 and it contains two subunits of Mr 16,000 that are not connected by disulfide bonds. The rat liver and the rat and human white blood cell racemase are immunologically related. They are completely inactivated by EDTA and can be activated by the addition of Co+2, with 50% activation occurring at a concentration of 0.2 microM. Lower levels for maximal activation were obtained with higher concentrations of Co+3, Fe+2, and Mn+2. Other metals such as Zn+2, Cu+2, Cu+1, and Cd+2 completely inhibited racemase even in the presence of equal concentrations of Co+2. The purified racemase appears to bind 1 mol Co/mol subunit.     

Isolation and characterization of chromatin from Caulobacter crescentus

The subunit structure of Caulobacter crescentus chromatin has been proven by electron microscope studies. The use of EDTA-Na2 during the purification of the chromatin complex enhanced the removal of contaminating ribosomes and non-chromatin proteins. The preparation obtained by modified procedure contained RNA polymerase as one of the major proteins and three histone-like proteins (10 K, 17 K and a hitherto not described protein with mol. wt 14 K).     

Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.     

Isolation and characterization of 5'-nucleotidase inhibitor from rat liver.

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.     

Polyribonucleotide synthesis by subfractions of microsomes from rat liver

1. The microsome fraction of rat liver has been fractionated and the ability of the fractions to incorporate ribonucleotides into polyribonucleotides has been studied. Activity was found in the rough-surfaced vesicle (light) fraction and in the free-ribosome fraction and this latter activity has been examined. 2. The free-ribosome fraction contains ribosome monomers, dimers and trimers together with some higher oligomers and ferritin. In addition to catalysing the incorporation of ribonucleotides into acid-insoluble material it contains diesterase activity. It catalyses the incorporation of UMP from UTP, but not UDP, AMP from ATP and CMP from CTP into polyribonucleotide material, and for UTP the product appears to be a homopolymer not more than eight units long attached to the ends of primer polyribonucleotide strands. 3. The activity could not be removed from the free-ribosome fraction by washing or by isolation in the presence of ethylenediaminetetra-acetic acid. 4. Partially hydrolysed polyuridylic acid but not polyadenylic acid could serve as a primer for the incorporation of UMP, but some activity was always associated with an endogenous primer. 5. Analysis of RNA extracted from the free-ribosome fraction after incubation with [³H]UTP showed the presence of 28s, 18s, 5s and transfer RNA types, but no radioactivity was associated with any of these RNA fractions.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Nucleosome-histone acetyltransferase from rat liver chromatin

Histone acetyltransferase capable of acetylating histones in nucleosome was extracted from rat liver chromatin. The enzyme was partially purified by hydroxyapatite chromatography. The enzyme preparation did not contain histones and required acceptors for enzyme reaction. The enzyme catalyzed acetylation of nucleosome-histones and free histones but did not catalyzed acetylation of DNA-histone mixture. Autoradiographic analysis revealed that the H4 was dominantly acetylated and other nucleosomal histones were also acetylated.