Sequestration of adenosine in crude extract from mouse liver and other tissues

Adenosine (1 μM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Sæbø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333–340). These data point to a role of this protein in the sequestration of adenosine in crude extract.The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed.The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 μM.The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract.The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.     

Activation of guanylate cyclase from rat liver and other tissues by sodium azide.

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

The availability of carnitine acetyltransferase in mitochondria from guinea-pig liver and other tissues

The carnitine acetyltransferase and glutamate dehydrogenase activities of guinea-pig liver and other tissues were estimated. Both enzymes are wholly mitochondrial, and can only be fully observed after disruption of the mitochondrion. Triton X-100 (0.1%) or freeze-drying revealed more activity than other methods tried. In mitochondria prepared and suspended in 0.25m-sucrose and in cell cytoplasm only small fractions of the total enzymic activity could be observed in guinea-pig liver: on average 7.5% of carnitine acetyltransferase and 5.5% of glutamate dehydrogenase. It is concluded that, in liver or mammary gland of goat, guinea pig or rat, little or no carnitine acetyltransferase is available in vivo to acetyl-CoA outside the mitochondrion.     

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Presence of D-aspartate oxidase in rat liver and mouse tissues

Rat liver D-aspartate oxidase activity, which had been reported to be undetectable, was found to be well detectable in dialyzed liver homogenate. The requirements of the enzyme for activity and its sensitivity to inhibitors were identical with the known properties of the enzyme from other sources. We also demonstrated for the first time the presence of the enzyme activity in mouse tissues and some other rat tissues using dialyzed tissue homogenates.     

Ascaris suum: suppression of reaginic and hemagglutinating antibody responses in the mouse by crude extract and maintenance fluid.

Intraperitoneal administration in mice of crude extract (CE) or maintenance fluid (MF) of Ascaris suum in Freund's incomplete adjuvant (FICA) in doses of 200 and 2 (CE) and 4 μg (MF) on Days ?4, 0, and +4 relative to the day of the immunization with 10 μg of hen egg white lysozyme (HL) resulted in the suppression of anti-HL reaginic antibody responses at varying degrees depending on the dose and their time of administration. Hemagglutinating antibody responses were also affected but in a different manner. Treatment with CE on Day ?4 resulted in complete suppression of reaginic antibody responses and some degree of suppression of hemagglutinating antibody responses depending on the size of the CE dose. In mice pretreated with MF, transient suppression was found only for reaginic antibody responses. In mice receiving the treatment of CE on Day 0, 200 μg of CE caused complete suppression of reaginic antibody responses, while 2 μg was less effective. Hemagglutinating antibody responses were also suppressed in proportion to the dose. Simultaneous treatment with MF did not cause any suppression of either reaginic or hemagglutinating antibody responses. In mice treated with CE on Day +4, reaginic antibody responses were not markedly suppressed and hemagglutinating antibody responses were also not altered. In contrast, treatment with MF on Day +4 resulted in suppression of reaginic antibody responses during the whole course of the primary response, but had no effect on hemagglutinating antibody responses. When MF was administered 7 days after the priming, no suppressive effect on the antibody responses was demonstrated. On the other hand, if a lower dose (1 μg of HL) was used for the priming, the effect of MF treatment with Day +4 was more pronounced in the primary reaginic antibody response and the secondary response was also affected. A comparable suppression of hemagglutinating antibody responses also was observed.     

Pilot-scale extraction and antimicrobial activity of crude extract from ovine neutrophils

Antimicrobial peptides have potential to be a high-value product purified from waste ovine blood. Previous work characterised antimicrobial peptides isolated from ovine neutrophils and determined the mechanisms of action. Here it is shown that the crude antimicrobial extract can be produced on a pilot-scale while retaining the antimicrobial activity. This crude extract could be used as a biopreservative for chilled lamb meat products or in a topical cream for treating cuts and grazes.     

A protein nuclear extract from D. melanogaster larval tissues

Preparation of protein nuclear extracts is often the first step to study in vitro biological processes occurring in the nucleus of the eukaryotic cell. Nuclear extracts have been extensively used in different model organisms to identify and study protein function in nuclei. Drosophila embryos can be collected in large quantities and have been the source of choice for the production of protein nuclear extracts. However, most of Drosophila in vivo studies on protein function are conducted in larval tissues. Here we report a new method to produce highly stable large-scale protein nuclear extracts from whole Drosophila larvae that are suited for a variety of biochemical analyses.     

Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of types 1 and 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2612 proteins having at least two unique peptides per protein. The data set also identified approximately 60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic data sets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at (http://ncrr.pnl.gov), provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields.     

Developmental changes of adenosine deaminase, xanthine oxidase, and uricase in mouse tissues

The activities of adenosine deaminase, xanthine oxidase, and uricase were followed in the liver, kidney, stomach, and intestine during pre- and postnatal development of the mouse. Results indicated that some type of coordinate control exists between the uricase and the xanthine oxidase levels in liver, stomach, intestine, and kidney. No coordinate control was seen between adenosine deaminase and xanthine oxidase in liver and kidney. The developmental changes between the intestine and stomach xanthine oxidase and adenosine deaminase were found to be related. The results obtained were consistent with the idea that intermediate metabolites in a pathway play some role in controlling the level of enzymes further down the pathway.A superificial resemblance in the timing of changes in feeding habits and changes in enzyme levels during development was found. Results of artifical change of feeding habit indicate that the control of enzyme levels was inherent rather than the result of dietary change.