Mouse Liver Extracts Research Articles

PH-Sensitive control of arginase by Mn(II) ions at submicromolar concentrations

The manganese dependence of arginase was reinvestigated with extracts of mouse liver to see whether more physiological properties were displayed than have been reported for the purified enzyme. In a preincubation with Mn(II) ions at 37 degrees C the enzyme underwent a slow and reversible activation. At least 90-95% of the activation achieved was dependent on Mn2+. However, no Mn2+ was required for catalytic activity in the assay. The activation showed little dependence upon pH over the range 6.5-9.5, whereas the catalytic activity increased 12-fold in apparent accord with the titration curve of an ionizable group of pKa 7.9. The Mn2+ dependence of arginase activation obeyed Michaelis-Menten kinetics, with Kd varying from 0.3 microM at pH 6.8 to 0.08 microns at pH 7.7. Free Mn2+ concentrations were established in these assays with a trimethylenediaminetetraacetate-Mn buffer. Vmax increased about three-fold over this range. The calculated arginase activity at 0.05 microM Mn2+ increases about nine-fold over this physiological pH range. An enzyme model is proposed to explain these findings. The activity of arginase at "physiological" [Mn2+] and the pronounced pH dependence conferred upon it are consistent with a recently revised role for the urea cycle in the control of bicarbonate and pH in the body. It appears possible that arginase loses Mn2+ sensitivity during the usual purification.

Immunopathological and Immunochemical Analysis of Autoimmune Enterocolitis in Mice

BALB/c (H-2d) and C57BL/6 (H-2b) mice were immunized with one or two doses of allogeneic or syngeneic mucosal antigen (MA) from small intestinal mucosa. This antigen was also characterized by immunoenzymatic assay (ELISA), by SDS-PAGE and by Western blotting. Single-dose immunized mice of either strain, sacrificed 30 days after immunization, synthesized marginal levels of anti-MA antibodies, as assessed by ELISA tests. BALB/c mice receiving two doses of the immunogen produced antibodies at a highly significant level (p less than 0.001) when compared to C57BL/6 mice belonging to the same experimental group. BALB/c mice immunized with syngeneic MA gave a humoral anti-MA response similar to that of BALB/c immunized with allogeneic MA. All experimental mice, irrespective of their genetic background or the number of doses of MA received, develop a cell-mediated immune response to MA. BALB/c mice immunized with two doses of allogeneic or syngeneic MA developed lesions mainly located in the small intestine, characterized by macroscopical and microscopical vascular congestion and hemorrhaging, epithelial cell loss, mononuclear cell infiltration of the lamina propria, cryptal degeneration and granuloma formation. Immunochemical analysis showed varying degrees of cross-reactivity between MA, liver and kidney saline extracts when heterologous anti-MA sera were tested by ELISA. Absorption of these sera with mouse liver or kidney saline extract lyophilizates reduced reactivity of anti-MA antibodies, although the degree of residual activity remained high. SDS-PAGE of mouse MA, kidney, and liver extracts revealed the presence of two polypeptidic bands of less than 17 kD molecular weight belonging to MA. Immunoblotting (Western blot) analysis revealed that heterologous or isologous anti-MA antibodies incubated with mouse MA reacted with the same epitopes as well as with others shared by other organs. Absorption of these sera with liver or kidney saline extracts revealed two organ-specific epitopes belonging to MA. Data presented here support the possibility of a genetic control of the susceptibility of BALB/c mice to the development of an acute autoimmune enterocolitis (AEC). This response appears to be a function of the amount of immunogen administered as well as the genetic background of immunized mice. The presence of a cell-mediated immune response to MA alone, however, is not sufficient for lesions to appear the histopathological alterations characteristic of AEC are present only in experimental groups where detectable levels of anti-MA antibodies are found. The antibody response seems to be directed against two unique epitopes belonging to MA.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative study of the action of C57BL and CBA mouse liver extracts on hepatocyte adhesion

Comparative study of the action of C57BL and CBA mouse liver extracts on hepatocyte adhesion

Translational inhibition by eIF-2-phospholipid complex in mammalian cell-free systems

The polypeptide chain initiation factor 2 (eIF-2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin-supplemented reticulocyte lysates [De Haro et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6711-6715]. This binding is independent of calcium ions and seems to be specific for phosphatidylinositol or phosphatidylserine; phosphatidic and arachidonic acids are inactive. Like alpha-subunit-phosphorylated eIF-2, eIF-2.PL traps GEF in a non-dissociable eIF-2.PL.GEF complex whereby GEF is no longer able to recycle. Initiation is inhibited when no free GEF is available. Translational inhibition by eIF-2.PL is rescued by equimolar amounts of eIF-2.GEF. On the basis of this stoichiometry, we have estimated that reticulocyte lysates contain about 60 pmol of GEF/ml (60 nM). eIF-2.PL also inhibits translation in cell-free mouse liver extracts and this inhibition is prevented by reticulocyte eIF-2.GEF suggesting that GEF also functions in liver. However, the eIF-2.PL complex does not affect translation in such non-mammalian eukaryotic systems as wheat germ and Drosophila embryos.

Purification and analysis of murine 2-5A-dependent RNase.

2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.

D-Amino acid oxidase in mouse liver

1. An appreciable amount of D-amino acid oxidase was found in the extract of mouse liver by enzyme-linked immunosorbent assay (ELISA). 2. The content of the enzyme in the kidney and heart extracts was also measured by the assay.

D-Amino acid oxidase in mouse liver—II

1. Activity of D-amino acid oxidase was detected in tissue extract of mouse liver by two sensitive spectrophotometric methods. 2. The activity was also detectable in extracts of the heart, but not of lung.

Effect of particle size of activated charcoal on separation of Triton X-100 from protein, liver cytosol, and lipoxygenase extracts

Triton X-100 was almost completely removed from bovine serum albumin solutions, BALB/c mouse liver extracts, and avocado peel lipoxygenase extracts by stirring the samples for 30 min in the presence of 250-350 mesh activated charcoal. The procedure did not remove protein significantly and did not reduce enzyme activity. At higher charcoal particle sizes, the efficiency of Triton adsorption was decreased and protein adsorption was increased. ElevateD temperatures enhanced Triton and protein adsorption. Adsorption on activated charcoal of 250-350 mesh is a simple and rapid procedure for Triton removal at a ratio of 0.23 g Triton X-100 per gram of activated charcoal.

Electrophoretic analysis of liver neuraminidase-1 variation in mice and additional evidence concerning the location of NEU-1.

Neuraminidase-1 (NEU-1) is one of two neuraminidase isozymes which can be detected electrophoretically in mouse liver extracts. The inheritance of variation in NEU-1 and the linkage relationships of the gene controlling this variation were studied through a backcross analysis involving the SM/J and MA/MyJ inbred strains, and by examination of NEU-1 phenotypes in three congenic strains: B10.SM, B10.SM(22R) and B10.RVB. The data indicate that NEU-1 is controlled by Neu-1, a gene previously identified by its effect on total liver neuraminidase activity in whole tissue homogenates. Analysis of the congenic strains revealed identical low activity (SM/J-type: Neu-1a/Neu-1a) NEU-1 phenotypes in all three strains. This indicates that Neu-1 lies in the segment of the SM/J-derived H-2 region that is common to all three strains: H-2E alpha to H-2D. In addition, we examined the relationship between NEU-1 and phenotypic variation in liver acid phosphatase (AP; for which a new typing method is described) and linkage order among several other enzyme-coding genes linked to H-2. In all animals that could be scored confidently for AP, the NEU-1 and AP phenotypes were concordant, adding support to the hypothesis that both phenotypes are controlled by Neu-1. Recombination rates among six H-2-linked marker loci were unexpectedly low, but were sufficient to verify the position of Upg-1 as the telomeric flanking marker relative to Glo-1, H-2 (C4), Neu-1 (Apl), Ce-2 and Pgk-2.

The diurnal rhythm of liver glycogen phosphorylase: correlating changes in enzyme activity and enzymic protein

The diurnal rhythm of phosphorylase activity in mouse liver extracts was correlated with the 24 h fluctuations in phosphorylase protein. This last measurement was made using rocket immunoelectrophoresis. The peak activity of phosphorylase appeared coincident (at 18:00 h) with the greatest amount of phosphorylase protein detected. Conversely, the lowest activity measured and lowest enzymic protein content both occurred at 02:00 h. Regression analysis revealed a significant positive correlation between enzyme activity and protein. Thus changes in the cellular concentration of this enzyme are implicated in the diurnal rhythm of liver glycogen.

Effect of hemolymph and nervous-tissue components on the in vitro development of flight-muscle myoblasts of Manduca sexta

Flight-muscle myoblasts from pupae of Manduca sexta were grown in the presence of either hemolymph or nerve extract. The cells exhibited distinctly different growth patterns: the addition of hemolymph yielded a branched network consisting of short, thin myofibers, while the addition of nerve extract resulted in parallel, extended fiber arrays. Spontaneous contractions were frequently observed. Other medium supplements (muscle extract, mouse-liver extract, β-ecdysone) had no detectable effect on myoblast development. Electron micrographs of explants grown in hemolymph revealed the presence of only a few myofibrils with a parallel filament arrangement but without cross striation, whereas explants grown with nerve extract exhibited cross striation within the voluminous filament arrays.

The regulation of cytosolic epoxide hydrolase in mice

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.

Analysis of neuraminidase isozyme phenotypes in mammalian tissues: An electrophoretic approach

A simple cellulose acetate electrophoretic method for visualizing mammalian neuraminidase isozymes has been developed. Application of the method with rat and mouse liver extracts reveals the presence of two distinct isozymes in each species. Each isozyme exhibits tremendous variation in activity between inbred strains. The two isozymes vary independently of one another suggesting that their activities are controlled by different genes. The neuraminidase phenotypes detected in these inbred strains via electrophoresis are consistent with published accounts of neuraminidase phenotypes determined fluorometrically in whole liver homogenates, but also indicate the presence of a second isozyme not perceived by this other procedure.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.

Protection of cultured mammalian cells by S-3-(3-aminopropylamino)ethylphosphorothioic acid(WR-2721)

The ability of WR-2721 to protect cultured mammalian cells against radiation-induced killing was nearly the same as that of cysteamine when WR-2721 was activated by mouse liver extract. Without the liver extract, protection by WR-2721 required long incubations with the cells prior to irradiation. The protective activity increased in proportion to the cell concentration. The dose reduction factor at a concentration of 4 mM WR-2721 was 1.11 and 1.41 for 1.5 X 10(5) cells/ml and 15 X 10(5) cells/ml of cultured cells, respectively. A non-protein bound sulfhydryl group was detected in cell suspensions after incubation with WR-2721, but it was not a dephosphorylated product of WR-2721.

Reduction of mitosis by an aqueous extract of uterine epithelium

Abstract Aqueous extracts of mouse uterine luminal epithelium have been shown to contain a factor which reduces mitosis in oestrogen-stimulated uterine epithelium, but not in vaginal epithelium or regenerating liver. The effect is transient and non-toxic, and occurs 3–4 hr after injection of the extract, with recovery by 6 hr, suggesting that there is a delaying action on cells in, or about to enter the G 2 phase of the cell cycle. This reduction in mitosis is not seen in response to aqueous extracts of mouse liver, or foreign proteins. Comparison of extracts prepared from uterine epithelia with high or low levels of proliferation, showed no difference in effectiveness.

Loss of the polyadenylate segment from mammalian messenger RNA: Selective cleavage of this sequence from polyribosomes

Abstract Treatment of mouse sarcoma 180 ascites cell polysomes with low levels of micrococcal nuclease, under conditions that cause relatively little fragmentation of the messenger RNA chains, results in considerable loss of poly(A) from these chains. This treatment generates a substantial amount of functional poly(A)-lacking mRNA. Brief incubation of cytoplasmic extracts of the ascites cells, and of mouse liver extracts, has similar effects on the polysomes present in the extracts and on the generation of poly(A)-lacking mRNA chains. The poly(A) segment is released from the polysomes treated with micrococcal nuclease as a nucleoprotein complex, and is protected from the action of the enzyme because of its association with protein. There is considerable poly(A) hydrolysis in incubated ascites cell extracts, and accumulation of a poly(A)-protein complex does not take place in this case. The liver extracts have little poly(A)-hydrolyzing activity, and free poly(A)-protein complexes are observed in these extracts. The poly(A)-cleavage process shows evidence of considerable selectivity. The newly synthesized mRNA population is more susceptible to this process than is the steady-state population. Moreover, only a portion of the steady-state mRNA loses its poly(A) readily upon incubation with micrococcal nuclease. Two-dimensional gel electrophoresis of translation products from total and poly(A)-lacking polysomal RNA preparations shows that not all mRNA species lose their poly(A) upon incubation of polysomes in ascites cell extracts. The sensitive population resembles the normal population of translatable poly (A)-lacking mRNA that is obtained from untreated polysomes. Individual species within this population show wide differences in their degree of susceptibility to the poly(A)-release process in vitro. Analysis by one-dimensional gel electrophoresis indicates that the same general population is generated by the incubation of cytoplasmic extracts and by the treatment of polysomes with micrococcal nuclease. It is suggested that the 3′ non-coding region of mRNA in polysomes is particularly sensitive to endonucleolytic cleavage, and that loss of poly(A) via this cleavage may be a normal cellular process. The diversity in nucleotide sequence and in overall configuration in this region could provide a basis for the observed differences in susceptibility to cleavage by nucleases.

A centrifugal column assay for thymidylate synthetase using the active site titrant 5-fluoro-2′-deoxyuridylate

Abstract Thymidylate synthetase has been titrated in mouse liver extracts with [ 3 H]fluorodeoxy-uridine monophosphate using centrifugally eluted gel filtration columns. The method is quick, efficient, and yields a linear dependence upon added extracted protein. The stability of the complex showed a pH maximum near 7.5. Specific binding of the tritiated nucleotide derivative to thymidylate synthetase was shown by denaturation of the complex in sodium dodecyl sulfate and gel electrophoresis of the denatured complex. Centrifugal elution of the sodium dodecyl sulfate-quenched complex from gel filtration columns was used to estimate the kinetics of complex formation at 0 and 12°C.

Purification of dihydrofolate reductase from human leukocytes and from mouse liver and electrofocusing on thin-layer polyacrylamide gel.

A method for the purification of dihydrofolate reductase starting upon such small quantities as 0.05--0.3 U is described. By ammonium sulfate precipitation, gel filtration and affinity chromatography on folate-aminohexyl-Sepharose extracts of leukocytes and mouse liver could be purified 1,500 and 830-fold, respectively. Electrofocusing on thin-layer polyacrylamide gels revealed two isoenzymes of mouse liver dihydrofolate reductase with pI values of 8.65 and 8.15. The purified leukocyte enzyme showed one band at pI 7.5. Addition by dihydrofolic acid or methotrexate prior to the isoelectric focusing did not change the behavior of the enzymes. Only addition of NADPH caused a slight decrease of the liver pI 8.15 and of the leukocyte band and the appearance of a faint band at pI 5.4 and 5.0, respectively.