Xg Supernatant Research Articles

Expression of an angiotensin‐(1‐7) endopeptidase in proximal tubules of the sheep and human kidney (1088.10)

Angiotensin‐(1‐7) [Ang 7] is a an active peptide of the renin‐angiotensin system (RAS) that generally antagonizes the actions of Ang II. Within the kidney, Ang 7 is derived from Ang I by endopeptidases including neprilysin (NEP) and thimet oligopeptidase (TOP) or from Ang II by the carboxypeptidase ACE2. Ang 7 is primarily degraded by ACE to the Ang‐(1‐5) in the circulation and tissues; however, we identified an endopeptidase in sheep CSF that efficiently degraded Ang 7 to Ang‐(1‐4) (Ang 4) and accounted for the majority of Ang 7 metabolism in this compartment (Marshall et al AJP 2013). The current study determined whether the Ang 7 peptidase is expressed in the kidney, specifically in isolated proximal tubules of the sheep and human renal epithelial cells. Activity was determined by the conversion of 125I‐Ang 7 to 125I‐Ang 4 in a 100,000 xg supernatant fraction. Both the sheep proximal tubules and the human epithelial cells expressed Ang 7 endopeptidase activity. Kinetic analysis revealed an apparent Km of 2.0 ± 1.0 µM and apparent Vmax’ of 93 ± 37 pmol/min/mg protein [n=3]. Peptidase activity in the isolated tubules was attenuated by the metalloendopeptidase inhibitor JMV‐390 with an IC50 of 0.81 ± 0.12 nM [n=3] that is ~50‐fold more potent than that of NEP and TOP. We conclude that an Ang 7 endopeptidase may be a novel component of the intrarenal RAS to regulate the tubular expression of Ang 7.Grant Funding Source: Supported by NIH grants HL56973, HL52972, HL112237, and HD047584

TAMM-HORSFALL PROTEIN AND URINARY EXOSOME ISOLATION: PP.9.380

Urinary exosomes have been proposed as a starting material for discovery of protein biomarkers of disease. Currently, standard protocols for urinary exosome isolation involve a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the 17,000 Xg supernatant and to sediment only when the sample is spun at 200,000 Xg. Here we show, using immunoblotting and electron microscopy, that urinary exosomes are also present in the 17,000 Xg pellet as a result of entrainment by polymeric Tamm-Horsfall protein (THP). This diminishes the reproducibility of isolation. We show, by electron microscopy, that the addition of DTT to the 17,000 Xg pellet results in disruption of the THP polymeric network presumably by reduction of disulfide bonds that link the monomers. This procedure was shown to deplete the exosomal proteins Alix, TSG101, and CD9 from the 17,000 Xg pellet. Also, by shifting the THP to the 200,000 Xg pellet, the use of DTT makes it theoretically feasible to use THP in this fraction to normalize excretion rates of other proteins in spot urines. Immunoblotting to test this idea showed a high degree of correlation between exosomal proteins and THP. We conclude that 1) THP polymeric networks entrain urinary exosomes; 2) the yield of exosomes by differential centrifugation can be increased by chemical reduction of the sample; and 3) THP may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical. Optimize urinary exosome isolation is a key point in the field of biomarker discovery

Characterization of Steroid Receptors in Human Ovarian Cancer

Steroid receptors for estrogen, progestogen and androgen in human ovarian cancer were characterized. 159,800xg supernatant was used as cytosol. In the cytosol, sucrose density gradients showed 5S and 8S binding peaks for each receptor. Steroid specificity studies showed that estrogen for [3H]-estradiol-receptor binding, progestogen for [3H]-R5020-receptor binding and androgen for [3H]-DHT-receptor binding were specific inhibitors. Equilibrium studies showed that the dissociation constant(Kd) was (15.3±3.1)x10-10M(n=8) for [3H]-estradiol receptor binding, (2.3, 4.6)x10-10M(n=2) for [3H]-R5020 receptor binding and (6.6, 26.0)x10-10M(n=2) for [3H]-DHT receptor binding.

Synthesis and Metabolism of 16α-Hydroxy-Steroids and Corticosteroids in Fetuses and Neonates

This study involved the biosynthesis and metabolism of a series of 16α-hydroxy-steroids and gluco- and mineralocorticoids in the fetus and their concentrations in materno-fetal plasma and amniotic fluid. In the biosynthesis of 16α-hydroxy-steroids, 17α-hydroxylase and C17–20-lyase in the adrenals and 16α-hydroxylase in the liver which are distributed in the microsomal fractions, extremely high activities are observed in 16–20 week fetuses. High sulfokinase activity is also found in the 105,000xg supernatant fraction and the activity in decreasing order is adrenal, liver and small intestine. These enzyme activities are affected by some steroids present in the umbilical plasma. In the biosynthesis of gluco- and mineralocorticoids, 17α-, 21-hydroxylase are distributed in the extramitochondrial fraction of the definitive adrenal cortex while 11β-, 18-hydroxylase and 18-dehydrogenase are in the mitochondrial fraction. All of these enzymes show high activities relatively early in gestation and angiotensin-II shows a selective stimulation for 11β-, 18-hydroxylase and potassium together with ACTH for 18-dehydrogenase. All of the 16α-hydroxy-steroids are consistently higher in the fetal plasma than in maternal plasma and show a 2 to 4 fold increase during the 3rd trimester of gestation. All of corticosteroids assayed are also higher in the fetal plasma and increase during this period. It is well established that a portion of cortisol undergoes sulfate conjugation or conversion to tetrahydro-derivative and that aldosterone is largely converted to 3α' 5β-tetrahydro-aldosterone and a portion undergoes glucosiduronation. Thus, in the fetus, some trophic hormones and endogenous steroids become active in the control of 16α-hydroxy-steroid and corticosteroid biosynthesis and metabolism. In conjunction, the presence of protein bound, unbound fractions as well as sulfate or glucuronide conjugated, unconjugated fractions in the blood tends to maintain an equilibrium and control over these systems.

Differential subcellular distribution and colocalization of microsomal and soluble epoxide hydrolases in rat cortical astrocytes.

The purpose of this study was to determine the expression, localization and subcellular distribution of microsomal (mEH) and soluble (sEH) epoxide hydrolases in cultured rat cortical astrocytes. Cultures of astrocytes were probed for mEH and sEH by immunocytochemistry, immunoblotting and enzymatic assays. Our results showed immunofluorescence for mEH and sEH, which colocalized with the astroglial cytoskeletal marker, glial fibrillary acidic protein (GFAP). Western blotting analyses for mEH revealed a protein band of molecular mass ∼50 kDa, the intensity of which was increased about 1.5‐fold, especially in the microsomal fraction. Whereas the polyclonal sEH rabbit serum recognized a protein band of molecular mass ∼62 kDa, which increased 2.0 fold in the 105,000xg supernatant over the cell lysate. The sEH rabbit serum also recognized similar protein bands in mitochondrial and nuclear fractions. The enzymatic analyses, using mEH‐ and sEH‐selective substrates further corroborated these findings. Our results suggest that astroglial cells, by expressing both mEH and sEH, may play a role in the cerebrovascular functions affected by epoxides (Funding Support: G12RR03045‐17 (ACS) and R37ES02710 (BDH)).

Influence of hypoxia and hypoxia/hypercapnia upon brain and blood peroxidative and glutathione status in normal weight and growth-restricted newborn piglets

Glutathione (reduced (GSH) and oxidized (GSSG)), lipid peroxidation products (TBAR) and in vitro production of reactive oxygen species (ROS, by means of stimulated lipid peroxidation, H2O2 formation and amplified chemiluminescence (CL) in 9000 xg brain supernatants) were studied in the cerebellum (C) and temporoparietal area (TP) of the brain of normal weight (NW) and spontaneously intra-uterine growth-restricted newborn piglets (IUGR) after 1 hour hypoxia (fractional inspired oxygen concentration (FiO2) 8%), and in combination with 10% CO2, followed by 3 hours recovery (FiO2 30%). The strong GSH depletion accompanied by an increased concentration of GSSG and TBAR, more distinct in IUGR, is the most important result in the brain after hypoxia and reoxygenation. Hypercapnia-related acidosis seems to protect the brain of IUGR from hypoxia/reoxygenation induced injury by reducing GSH depletion as well as GSSG and TBAR increases. But stimulated lipid peroxidation and H2O2 formation in 9000 xg supernatants of C and TP were found to be higher in acidosis and hypercapnia. Decreased or unchanged amplified CL, demonstrating lower in vitro production of ROS, cannot explain the GSH depletion after hypoxia and reoxygenation. The scarce changes in erythrocyte GSH and GSSG as well as plasma TBAR concentrations did not reflect the findings in the brain. Nevertheless, the changes in the brain support the hypothesis that oxidative stress plays a role in neuronal damage after hypoxic stress, but the brain of IUGR did not reveal a special response to moderate hypoxia.

Effect of soil ingestion on the storage of Se, vitamin B12, Cu, Cd, Fe, Mn, and Zn in the liver of sheep fed lucerne pellets

Abstract The effect of ingesting 100 g/day of two yellow‐brown/yellow‐grey earth soils on the storage of selenium (Se), vitamin B12, copper (Cu), cadmium (Cd), iron (Fe), manganese (Mn), and zinc (Zn) in the liver of sheep was studied in 32 wether lambs fed lucerne pellets. Both soils significantly increased plasma and liver concentrations of Se. Soil B which had the highest cobalt (Co) concentration (1.8 versus 1.32 ug/g DM) significantly increased the liver vitamin B12 concentration. The soils did not affect the concentrations of Cu, Mn, Fe, and Zn in the liver. An increase in liver Cd concentrations occurred during the study but this was not related to the soil treatments. Soil ingestion increased in the 30 000 xg supernatant fractions Fe concentrations in all digesta, Mn and Se concentrations in the abomasal digesta, Se and Zn concentrations in the reticu‐lorumen, and decreased Cu concentrations in the abomasal digesta. Depending on the amounts ingested, the yellow‐brown/yellow‐grey earth soils could ...

Biotransformation of Phenyl- and Pyridylalkane Derivatives in Rat Liver 9,000xg Supernatant (S-9)

Biotransformation of Phenyl- and Pyridylalkane Derivatives in Rat Liver 9,000xg Supernatant (S-9)

Simple method of RNA isolation from human leucocytic cell lines.

Very reliable methods have been developed to extract RNA from different tissues and cell lines (1, 2, 3). RNA preparation, however, is still cumbersome and time consuming, especially when extracting a large number of cells (>107) and cell samples. The isolation of RNA from leucocytic cells is particularly difficult for three reasons: a) high levels of endogenous RNase; b) low amount of RNA (RNA/DNA ratio of 0.08 in granulocytes compared to 4.0 in cultured fibroblasts) (3); c) contamination of the RNA isolate with DNA and protein. To overcome these problems, we developed a simple and efficient RNA isolation procedure by modifying the method described by Chomczynski and Sacchi (2). Our modified protocol has the following advantages: a) it is simple to perform and needs no major equipment; b) it reduces drastically the volume of costly and hazardous solvents; c) both, small scale (5 x 106 cells: RNA yield 30-50 jig) and large scale (1 x 108 cells: RNA yield 300-600 ILg) RNA extractions can be performed using exactly the same protocol in small 1.5 ml microfuge tubes with many cell samples (up to 20) in parallel in a short time (5 hours); d) the protocol produces extremely pure total RNA at high yield. We used the method mainly to measure quantitavely low level gene expression by Northern blot. The following detailed stepby-step protocol was tested using various undifferentiated and differentiated leucocytic cell lines (U937, HL60, HEL, MM6), but should be applicable to any cell type growing in suspension. Pellet leucocytic cells from a 50 ml culture flask (1x105-2x106 cells/ml) in a 50 ml Falcon tube. Pour off medium, turn Falcon tube upside down and put it on a kimwipe to drain the pellet. While vortexing the pellet, splash 600 1l solution D (4M guanidinium thiocyanate; 25 mM sodium citrate, pH 7; 0.5% sarcosyl; 0.1% 2-mercaptoethanol; 0.1% antifoam A) to the cells. Vortex until reaching a clear very viscous lysate. Pour the suspension directly into a 1.5 ml Eppendorf cup. Aspirate the lysate into a 2 ml syringe fitted with a 23G-needle and expel it into the tube applying high pressure. Repeat this step 10 times. (Antifoam A prevents foaming while shearing DNA). Add 600 y1 8 M LiCl, mix by turning the cup until the clear suspension becomes cloudy (do not vortex). Cool on ice for at least 1 hour. Spin down precipitated RNA at 13000Xg for 20 min. at 4°C. Remove completely supernatant. Take up RNA pellet in 650 ,I DEPC-treated water and transfer suspension to another tube. Then add 700 itl acidic phenol (pH 4.3) and vortex vigorously. Add 140 td chloroform/isoamyl-alcohol (24:1) and vortex vigorously. Spin tube (13000 g, 10 min., 4°C) and transfer 650 $1 from the aqueous upper phase into another tube and vortex vigorously with 650 Al chloroform/isoamyl alcohol (24:1). Spin again (13000 g, 10 min, 40C) and carefully recover 500 1d from the aqueous supernatant and transfer it into a new tube. Add 50 11 8 M LiCl, mix, add 500 A1 precooled (-200C) isopropanol and mix well (do not vortex). Precipitate RNA for 30 min. at -200C. Spin down RNA at 11000 xg, 10 min., 4C and remove supernatant completely. Rinse pellet with 1 ml 70% ethanol and spin again at 13000 xg, 5 min., 40C. Aspirate supernatant, dry the pellet only slightly and resuspend RNA in DEPC-treated water (100,ul). Measure O.D. and store RNA suspension at20°C. A wide range of cell numbers (5 x 106108 cells) was lysed in always the same volume of solution D (600 IAI), instead of adjusting the volume of solution D to the actual cell count (100 /d solution D per 106 cells) as indicated in (2). This procedure dramatically reduced the volume of solution D (up to 20-fold)

15-Ketoprostaglandin delta 13-reductase activity in bovine ocular tissues.

The present study provides the first evidence for delta 13-reduction of 15-ketoprostaglandins (15-keto-PGs) by bovine ocular tissues. The 9,000xg supernatants of cornea, iris, ciliary body, retina, and RPE-choroid except lens exhibited delta 13-reductase activity toward 15-keto-PG E2 and F2 alpha in the presence of NADPH or NADH as an electron donor. Among the tissues tested, the highest activity was observed in ciliary body and iris, followed by RPE-choroid, retina, and cornea. The NADPH- and NADH-linked double bond reductase activities were inhibited by dicumarol, quercitrin, indomethacin and disulfiram, but not by potassium cyanide. NADPH-linked 15-keto-PG F2 alpha delta 13-reductase was purified from bovine iris-ciliary body cytosol by fractionation with ammonium sulfate and high performance liquid chromatography (HPLC) with TSK gel DEAE-5PW, and TSK gel Blue-5PW. The purified enzyme was homogenous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular weight was estimated to be about 57,000 by electrophoresis, and about 55,000 by gel filtration HPLC with Superose 12.

Effects of phenobarbital and 3‐methylcholanthrene pretreatment on the pharmacokinetics and the pharmacodynamics of bumetanide in rats

The effects of pretreatment with the enzyme inducers, phenobarbital (PB) and 3-methylcholanthrene (3-MC), on the pharmacokinetic and pharmacodynamic parameters of bumetanide were examined in rats. The nonrenal clearance (19.3 vs 29.6 ml min-1 per kg) of bumetanide increased significantly in PB treated rats. This suggested that the nonrenal metabolism of bumetanide is increased by pretreatment with PB, which was supported by significantly increased amounts of bumetanide glucuronide and desbutyl bumetanide excreted in 8-h urine, and reduced amounts of bumetanide remaining per gram of tissue after 30-min incubation of 100 micrograms of bumetanide with the 9000 xg supernatant fraction of liver, stomach, and kidney tissue homogenates in PB treated rats. The contents of hepatic cytochrome P-450 (1.29 vs 2.15 nmol mg-1 protein) and the weights of liver and stomach increased significantly in PB treated rats, suggesting that the metabolizing enzymes for bumetanide are induced by pretreatment with PB. The 8-h urine output per 100 g body weight was not significantly different by pretreatment with PB although the amounts of bumetanide excreted in 8-h urine increased significantly in PB treated rats. It could be explained by the fact that the dose of bumetanide used results in urinary concentrations at the plateau of the concentration-effect relationship. Therefore, the alteration in the urinary excretion rate of bumetanide by pretreatment with PB would not alter the diuretic effect. In 3-MC treated rats, pharmacokinetic and pharmacodynamic parameters were not significantly different and it suggested that the metabolizing enzymes for bumetanide are not induced by pretreatment with 3-MC although the contents of hepatic cytochrome P-450 and the weights of liver and stomach increased significantly by pretreatment with 3-MC.

Identification of 12(S)-hydroxyeicosatetraenoic acid in the young rat lens

Evidence is presented indicating that young rat lens has the capacity to synthesize 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) from exogenous arachidonic acid (AA). A 9,000 xg supernatant prepared from 15 day old rat lenses, when incubated with calcium and U-[14C]-AA, generated a radiolabelled product with a retention time identical to authentic unlabelled 12-HETE in two different HPLC solvent systems. Mass spectral analysis provided evidence that the metabolite was 12-HETE while chiral studies demonstrated the exclusive presence of the 12(S) isomer. Radiolabelled 12-HETE synthesis was inhibited by preincubating the 15 day old rat lens supernatant with 0.2 Mm curcumin, a mixed cyclooxygenase/lipoxygenase inhibitor. Radiolabelled 12(S)-HETE synthetic capacity was highest in the 4 day old rat lens, the earliest time period measured, and rapidly declined with age reaching negligible levels at about 4 months. These results suggest that the young rat lens possess the biosynthetic capacity to generate radiolabelled 12(S)-HETE from U-[14]C-AA. The profile of 12-HETE synthetic activity suggests a possible regulatory function of the hydroxylated AA metabolite in the developing lens.

Enzymatic sulfation of gastrin in rat gastric mucosa

An enzyme which catalyzes the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a K m value of 5 μM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.

The induction of alkoxyresorufin metabolism: A potential indicator of environmental contamination

Methods of biochemical monitoring of individual animals for exposure to environmental contaminants are of great potential use. The hepatic metabolism of various alkoxyresorufins, which are highly specific substrates for certain forms of cytochrome(s) P450, is highly induced by a variety of environmental contaminants. Thus, the O-dealkylation of pentoxy- or benzyloxyresorufin was induced greater than 20-fold in the rat by alpha-hexachlorocyclohexane, 2,4,5,2',4',5'-hexabromobiphenyl, DDT and Aroclor-1254, while the metabolism of ethoxyresorufin was highly induced by 5,6-benzoflavone, 3,4,5,3',4',5'-hexabromobiphenyl and Aroclor-1254. Additionally, rats exposed to diets containing as little as 12 ppm DDT displayed greater than five-fold increases in the rate of hepatic O-dealkylation of benzyloxyresorufin. Induction of the hepatic metabolism of these resorufin ethers in 9000 xg supernatant fractions taken from rats exposed to potential environmental contaminants may constitute a valuable diagnostic indicator of the presence of a variety of pollutants, including polycyclic aromatic hydrocarbons, organochlorine pesticides, polyhalogenated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results suggest the potential applicability of these substrates in detecting chemical contamination in the environment.

Accumulation of unglycosylated liver secretory glycoproteins in the rough endoplasmic reticulum

Previous studies in this laboratory (1) have shown that tunicamycin-treatment inhibits the secretion of three secretory glycoproteins--alpha 2-macroglobulin, ceruloplasmin, and alpha 1-protease inhibitor in human hepatoma (Hep G2) cell cultures. In the present study, we have investigated (i) their site of accumulation within the endoplasmic reticulum/Golgi pathway, and (ii) the solubility characteristics of these unglycosylated proteins. Using percoll density gradient centrifugation, we found that tunicamycin-treatment markedly inhibited the transport of alpha 2-macroglobulin, ceruloplasmin and alpha 1-protease inhibitor from the rough endoplasmic reticulum. However, there was no detectable changes in their solubility properties as both the glycosylated and unglycosylated species were associated with the 100,000 xg supernatant fraction following disruption of the microsomal fraction (i) with 0.2% Triton X-100 and (ii) by repeated freeze-thaw cycles. Also no evidence of protein aggregation was detected by liquid chromatography of the unglycosylated proteins on Bio-Gel A-1.5 column.

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A 4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is >15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[ 14C]HPETE is generated from [ 14C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[ 14C]HETE produced is of much lower specific activity than the [ 14C]arachidonic acid. This indicates that the 5-[ 14C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.

53 LOCALIZATION OF XANTHINI DEHYDROGENASE IN LIVER

Xanthine dehydrogenase has been reported to be present in an endothelial cell in beef or rat liver and also in peroxisome of a hepatic cell. However, as the most enzyme was recovered in 100,000xg supernatant of rat or chicken liver homogenate, cellular and subcellular localization in liver are still obscure. We reexamined localization of xanthine dehydrogenase in the tissue of chicken liver by imrnunohistochemical method, or by the method of isolation of hepatic cells. Chicken liver xanthine dehydrogenase purified by the method of Nishino was used for the immunization of rabbit. The antisera obtained from the rabbit gave a distinct single precipitin line in agar against chicken liver xanthine dehydrogenase or crude extract. This antisera were used for immunohistochemical experiments. Chicken liver were perfused with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin and sectioned. They were stained with the indirect method using enzyme labeled antibody. The positive staining was observed in the cytoplasm of hepatocytes, Kupffer cells and endothelial cells, suggesting that these type cells contain xanthine dehydrogenase. After isolation of hepatocytes most of xanthine dehydrogenase was recovered in 100,000xg supernatant fraction from homogenate of hepatocytes and the specific activity of enzyme was similar to that in 100,000xg supernatant fraction from homogenate of liver.

182 PURIFICATION OF RAT LIVER AMIDOPHOSPHORIBOSYLTRANS FERASE (ATase)

Purification of mammalian ATase is essential for the understanding of the regulation of de novo purine synthesis in mammals. The basic properties of rat liver ATase after step 4 of purification methods described below includes a) feedback inhibition by adenosine or guanosine mono-, di- and tri-phosphates, with the decreasing order of magnitude, b) Inhibition by 1, 10-orthophenanthroline as a Fe++ chelator, but not by 1, 7-metaphenanthroline, and c) Inactivation by exposure to oxygen with a half life of 15 min at 37°C with the stable activity under argon. In this study ogygen inactivation of ATase was overcome by the use of oxygen-free buffer and HPLC methods. The purification procedures include the following 8 steps. 1. 55,000xg supernatant of rat liver homogenate, 2. Heat treatment to 56°C, 3. Acid precipitation to pH 5.1, 4. Ammonium sulfate fractionation between 30 and 55%, 5. Gel filtration by Toyopeari HW55F, 6. DEAE-5PW HPLC column, 7. Hydroxyapatite HPLC column, 8. SDS polyacrylamide gel and electroelution. After step 7, ATase was purified to 958-fold with 4.1% recovery. After step 8, ATase was finally purified to homogeneity proved by a single protein band with silver stain. The monomer size of ATase was 62K on SDS polyacrylamide gel. The amount of ATase in rat liver is estimated as about 10 μg/g liver. The methods of purification of mammalian ATase open the research direction to molecular cloning and monoclonal antibody production.

A Novel growth factor in rat spleen which promotes proliferation of hepatocytes in primary culture

We have identified a factor from adult rat spleen which stimulates the proliferation of rat hepatocytes. The activity was found in the spleen soluble matrix fraction (1,300xg supernatant of inter-cellular fraction). No activity was found in the spleen cell homogenate, in the spleen insoluble matrix fraction or rat serum. After 4 days of incubation with the spleen factor, the cell number increased 4-fold higher than that at inoculation. The growth stimulation were observed in both fetal bovine serum supplemented medium and hormonally defined medium which contains insulin, epidermal growth factor, glucagon, growth hormone and prolactin. The level of activity in the spleen soluble matrix was not affected by partial hepatectomy or trypsinization. These data indicate that the spleen factor is different from previously characterized effectors of hepatocyte proliferation. The novel factor has been named spleen derived growth factor (SDGF).

Comparison of some biochemical properties of epidermis in tumor promotion-susceptible and -resistant strains of mice.

It has been reported that CD-1 and SENCAR mice are susceptible and C57BL/6 mice are resistant to skin tumor promotion caused by phorbol esters. Specific binding of a phorbol ester to its epidermal receptor site, epidermal protein kinase C activity, and ornithine decarboxylase (ODC) induction in epidermis were compared between tumor promotion-susceptible and -resistant strains of mice. Specific binding of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to the particulate fraction of the epidermis of C57BL/6 mice gave a similar dissociation constant (Kd) and a maximal number of binding sites (Bmax) to those of CD-1 mice. Protein kinase C activity of the epidermal 105,000 xg supernatant was not significantly different between C57BL/6 and CD-1 mice. Protein kinase C activity of the 105,000 xg pellet, however, was significantly higher in C57BL/6 mice than in CD-1 mice. A topical application of TPA to the skin caused epidermal ODC induction in all of these strains of mice. At any doses of TPA, TPA-induced epidermal ODC activity of C57BL/6 mice was always higher than those of SENCAR and CD-1 mice. Maximal induction of epidermal ODC by TPA was also highest in C57BL/6 mice among these three strains of mice. These results indicate that the mechanism of the difference in susceptibility of C57BL/6, CD-1 and SENCAR mice to the tumor-promoting action TPA resides in a step distal to or other than the protein kinase C activation and ODC induction.