Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

Human galactose-1-phosphate uridylyltrsferase: purification and comparison of the red blood cell and placental enzymes

Galactose-1-phosphate Uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate Uridylyltransferase, EC 2.7.7.12) has been purified from human red blood cells and placental tissue. The placental enzyme was obtained as a homogeneous protein with a specific activity of about 100 units/mg of protein by a combination of previously published methods (G. R. Helmer, Jr., and V. P. Williams, 1981,Arch. Biochem. Biophys.210, 573–580) and concanavalin A-Sepharose chromatography. The properties of the two enzyme forms have been examined with respect to subunit size, electrophoretic properties, isozyme distribution, kinetic patterns, and immunological properties.     

A human lymphoid cell line with receptors for both sheep red blood cells and complement

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) EAC rosettes were formed more rapidly and were more stable than E rosettes; (b) preincubation of MOLT with an EAC membrane preparation inhibited resetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.     

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: changes during reticulocyte development and effects on the beta receptor

Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower (P < 0.01) than those concentrations of the factor from erythrocytes necessary to augment isoproterenol-responsive adenylate cyclase activity in reticulocyte (9.7 ± 2.3) and erythrocyte (7.5 ± 1.0) membranes. Cytosol factor from reticulocytes also caused greater total isoproterenol responsiveness than that from erythrocytes both in reticulocyte (784 ± 107 vs 525 ± 65 pmol/mg protein) and in erythrocyte membranes (54 ± 6 vs 36 ± 3); P < 0.05. Neither reticulocyte nor erythrocyte cytosol factor affected the concentration at which isoproterenol half-maximally stimulated adenylate cyclase in either set of membranes. However, the cytosol factor from reticulocytes markedly decreased the binding affinity of isoproterenol for β receptors in reticulocytes from 0.8 ± 0.2 to 6.9 ± 1.4 μm; P < 0.001. This reticulocyte factor had no significant effect on the binding affinity of isoproterenol for erythrocyte membranes. Erythrocyte factor did not change the binding affinity for isoproterenol in either reticulocyte or erythrocyte membranes.     

On the distribution of cell cycle generation times

The problem of whether the cell cycle is a deterministic or probabilistic process is widely discussed in the current literature (P. Nurse, Nature, 286, pp. 9–10, 1980). In this report the question of fluctuations of cell cycle period is treated in the limits of the membrane model of cell division regulation. The parametric analysis of the equations set both for normal and tumour cells is carried out. We describe the bifurcation parameters in the neighbourhood of which the system can amplify the small fluctuations. The presence of white noise in parameters describing the lipids and antioxidants influxes into membrane is examined by methods of Marcovian processes and also by direct stochastic computer simulation. The equation for the distribution function of generation times is obtained and the increase of dispersion and mean cycle time during the changes of those parameters which would be connected with cell culture density is calculated.The influence of parameter fluctuations upon the cycle period for both normal and tumour cells is compared in the framework of model assumptions. The ratio of dispersion of generation time distribution to mean period value for an ensemble of tumour cells is shown to be several times greater than that for normal ones.In the discussion the problem of the presence of a premitotical (G₀₂) resting state and of the possibility of its experimental detection is considered.     

Ethanol reduces hepatic estrogen-2-hydroxylase activity in the male rat

Brief exposure to intoxicating levels of ethanol in the male rat produced a marked reduction in a major hepatic enzyme responsible for estrogen metabolism (estrogen-2-hydroxylase). After 4 days of ethanol administration the specific activity of this enzyme decreased by 70% and remained decreased for 6 days following alcohol withdrawal. Enzyme activity returned to control levels by two weeks. However, if animals were retreated with ethanol for one day each week the enzyme activity remained low. Kinetic analysis of the enzymatic activity from ethanol-treated rats showed a decrease in specific activity (Vmax) with no alteration in substrate affinity (apparent Km). The decrease in enzyme activity persisted long after ethanol disappeared from the blood and concentrations of ethanol from 20–100 mM had no effect on enzyme activity when added $\text{in vitro}$. A similar effect of ethanol on hepatic estrogen metabolism in humans may partially explain the elevated serum estrogen levels and the signs of hyperestrogenization observed in male alcoholic patients.     

(Des-Histidine1) (Nϵ-phenylthiocarbamoyllysine12)-glucagon: Effects on glycogenolysis in perfused rat liver

(Des-Histidine¹) (N^?-phenylthiocarbamoyllysine¹²)-glucagon, synthesized by the one-step Edman degradation procedure is a competitive inhibitor of glucagon action in the rat liver plasma membrane adenylate cyclase system. However, in the perfused rat liver, the compound did not inhibit glucagon stimulated glycogenolysis even when used at a concentration 100-fold in excess of native glucagon. Instead, it showed a weak potency, but full agonist activity, stimulating liver glycogenolysis to 100% of the level obtained by glucagon. These results are discussed in terms of the possible mechanism(s) of glucagon action.     

Microdetermination of galactose and galactose 1-phosphate in dried blood spots

Newborn screening for galactosemia (galactose-1-phosphate uridyltransferase deficiency), as well as for other defects in galactose metabolism (galactokinase deficiency and uridine diphosphogalactose 4-epimerase deficiency), requires a method of determining both galactose and galactose 1-phosphate in dried blood. We have developed a sequential quantitative method for the microdetermination of galactose and galactose 1-phosphate that can be applied to 3-mm-diameter disks of dried blood and that can be used with a Technion Autoanalyser II equipped with a fluorometer.Galactose is determined by the fluorescence of NADH following treatment with β-galactose dehydrogenase and with the consequent reduction of NAD. The complete system includes alkaline phosphatase for the hydrolysis of galactose 1-phosphate, so that the total amounts of a galactose and galactose 1-phosphate are determined. For the measurement of galactose alone, alkaline phosphate is omitted from the system. The difference in fluorescence between that from the complete system and that from the alkaline phosphatase-omitted system yields the concentration of galactose 1-phosphate.     

Prostaglandin A1 and E1 inhibit the plating efficiency and proliferation of murine melanoma cells (Cloudman S-91) in soft agar

PGA₁ and PGE₁ reduced the plating efficiency and inhibited proliferation of Cloudman S-91 murine melanoma cells in a dose dependent manner, as assessed by their effects on colony formation in soft agar. PGF_2α did not reduce plating efficiency but was as effective as PGA₁ in raising cAMP and cGMP levels. This data suggests that the inhibition of Cloudman S-91 murine melanoma cell growth occurs via a non-cyclic nucleotide mechanism.     

In vitro induction of primary and secondary xenoimmune responses by liposomes containing human colon tumor cell antigens

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl₂ to a dried lipid film. Cytotoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposome-antigens also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposome-antigens were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens. Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select desired immune responses, as well as to identify colon tumor-specific determinants.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Effect of antinociceptive doses of oxotremorine on mouse brain acetylcholine turnover rate

The effect of antinociceptive doses of oxotremorine on the steady-state level and turnover rate of acetylcholine (ACh) was investigated in male Swiss-Webster mice. Oxotremorine produced dose-related increases in ACh levels which attained statistical significance (p ≥ 95; Dunnett's T) with the ED₈₄ antinociceptive dose in two sacrifice methods. The increased steady-state level of ACh was temporally correlated with the peak antinociceptive effect of oxotremorine. ACh turnover rate decreased with increasing doses of oxotremorine. The ACh turnover rate decreased from 11.06 ± 1.62 nmol/g/min to 5.38 ± 0.71 nmol/g/min by decapitation method and 30.20 ± 1.8 nmol/g/min to 19.99 ± 1.6 nmol/g/min by the microwave irradiation method (Ed₈₄ oxotremorine doses). The decrease in turnover rate of ACh produced by antinociceptive doses of oxotremorine is of a lesser magnitude than that produced by tremorogenic doses (1,2).     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Thermal inactivation of a Citrobacter ribonuclease: Influence of polyamines and ionic strength

The thermal inactivation of a Citrobacter sp. ribonuclease (RNase) is subject to control by a number of factors. Low concentrations of naturally occurring polyamines such as spermidine and spermine, and certain analogs of these compounds, protect the enzyme from inactivation. Changes in ionic strength cause wide variations in the rate at which enzyme activity is lost. Additionally, depending on the type of ion added to the reaction mixture, the rate constant for enzyme inactivation-may either increase or decrease as the ionic strength is raised. Thermodynamic parameters were determined under a variety of experimental conditions for the thermal inactivation of this RNase. It was found in all of these cases that the entropy of activation is large and negative, implying that a gross change in enzyme conformation is not taking place. The concentration and identity of ions present and the amount of polyamine available to interact with this RNase determines the rate of loss, by thermal inactivation, of enzyme activity in this in vitro system. These factors therefore constitute a system whereby substrate hydrolysis may be controlled with time.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

The effect of sodium ion on antinociception and opiate binding in vivo.

Large quantities of NaCl and CaCl₂ but not KCl given intrapertoneally decreased the antinociceptive activity of morphine. NaCl also antagonized the effect of morphine on the stereo-specific binding of opiates. This high dose of NaCl doubled the level of sodium in the brain but did not alter the specific gravity of brain tissue. These $\text{in}$$\text{vivo}$ effects of NaCl confirm the antagonistic effects of NaCl $\text{in}$$\text{vitro}$ that have been reported.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Metabolism of furazolidone by milk xanthine oxidase and rat liver 9000g supernatant: formation of a unique nitrofuran metabolite and an aminofuran derivative

In vitro metabolism of furazolidone (N-(5-nitro-2-furfuryliden)-3-amino-2-oxazolidone) was investigated by using milk xanthine oxidase and rat liver 9000g supernatant. As a result, a new type of reduction product was isolated as one of the main metabolites from the incubation mixture and it was tentatively identified as 2,3-dihydro-3-cyanomethyl-2-hydroxyl-5-nitro-1a, 2-di(2-oxo-oxazolidin-3-yl)iminomethyl-furo[2,3- b]furan. In addition, the present study demonstrated the formation of N-(5-amino-2-furfurylidene)-3-amino-2-oxazolidone as a minor metabolite of nitrofuran in a milk xanthine oxidase system. The aminofuran derivative was easily degraded by milk xanthine oxidase under aerobic, but not anaerobic, conditions. The degradation appears to be due to superoxide anion radicals, hydroxyl radicals, and/or singlet oxygen, which are produced in this enzyme system.     

Effect of corticosterone on the prolactin response to psychological and physical stress in rats

Both corticosterone and prolactin (PRL) levels increase in response to stress. In these studies we examined the effect of corticosterone on the PRL response to both physical (footshock) and psychological (novel environment) stress. Three groups of rats were used: sham adrenalectomized (SHAM), adrenalectomized (ADX), and adrenalectomized with corticosterone replacement (ADX+CORT). The corticosterone-treated animals received 80 ug corticosterone/ml drinking water. Blood samples were drawn via an indwelling cannula and PRL values determined using radioimmunoassay. ADX rats showed a consistently greater PRL response to being placed on a platform above water (novel environment) or when receiving intermittant footshock than did ADX+CORT rats. The PRL response of the latter group was similar to that of the SHAM animals. These findings indicate that corticosterone levels of an animal can significantly attenuate the magnitude of the PRL response to both physical and psychological stress. These findings further emphasize that the PRL response to stress is dependent not only upon the immediate action of the stressor, but also the prior stress history of the animal.