The comparative effects of 5′-methylthioadenosine and some of its analogs on cells containing,and deficient in, 5′-methylthioadenosine phosphorylase

The antiproliferative effects of 5′-methylthioadenosine and the 5′-methylthioadenosine analogs, 5′-isobutylthioadenosine, 5′-deoxyadenosine and 5′-methylthiotubercidin were examined using two mouse cell lines, one 5′-methylthioadenosine phosphorylase-deficient the other containing 5′-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5′-Methylthioadenosine, 5′-deoxyadenosine and 5′-isobutylthioadenosine, all of which are substrates for the 5′-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5′-methylthioadenosine phosphorylase-deficient cells than to the 5′-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5′-methylthiotubercidin, a nondegradable inhibitor of the 5′-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5′-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5′-methylthiotubercidin on 5′-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5′-methylthioadenosine was explored using both cell types. 5′-Methylthiotubercidin inhibited the accumulation of exogenous 5′-methylthioadenosine from 5′-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5′-methylthioadenosine. In contrast, 5′-methylthiotubercidin caused a large accumulation of extracellular 5′-methylthioadenosine with a concomitant smaller increase intracellularly in 5′-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5′-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5′-methylthiotubercidin suggests two possible sites at which 5′-methylthiotubercidin may exert its effect.     

Further characterization of L-β-hydroxyacid dehydrogenase from Drosophila

L-β-Hydroxyacid dehydrogenase (L-β-hydroxyacid--NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-β-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-β-hydroxyacid dehydrogenase IgG revealed a single L-β-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H⁺ ejection/2e^? ratio approaches an average value of 3 when K⁺ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ejection/2e^? ratio of 2 is observed. (3) The low stoichiometry of 3H⁺ ejected (instead of 4) per 2e^? and the high rate of H⁺ back-decay (0.1615 $\text{ln}\text{δ-}\text{(}\text{ngatom}\text{)}\text{H}{}^{\mathrm{+}}\text{s}$ and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K⁺/H⁺ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.     

Preliminary characterisation of inhibitors of DNA polymerase isolated from Xenopus laevis early embryos

Inhibitors of DNA polymerase have been detected in Xenopus laevis ovary and egg extracts. The characteristics of the inhibitors differ between the two extracts. In ovary preparations, the inhibitor is retained by dialysis tubing and is heat sensitive, whereas in egg extracts it is diffusable and heat stable. In both extracts, the activity co-elutes with DNA polymerase after ion exchange chromatography. Chromatography of ovary extracts renders the inhibitor diffusable and heat stable. Preliminary characterisation of inhibitory activity from eggs shows that the substance is sensitive to pronase digestion and has an approx. 300–500 molecular weight. Kinetic studies demonstrate that the inhibitor is uncompetitive with the DNA template and show mixed inhibitory kinetics with respect to the deoxynucleotides.     

Melanoprotein Absence of a direct melanin-protein relationship in chick embryp melanocytes

Short-term synthesis of radioactivity labeled melanin (using dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cyloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in l-[U-¹⁴C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Na+-Ca2+ exchange in the axolemma-rich membrane vesicle preparations from the walking-leg nerves of the american lobster

An axolemma-rich membrane vesicle fraction was prepared from the leg nerve of the lobster, Homerus americanus. In this preparation Ca²⁺ transport across the membrane was shown to require a Na⁺ gradient (Na⁺-Ca²⁺ exchange), and external K⁺ was found to facilitate this Na⁺-Ca²⁺ exchange activity. In addition, at high Ca²⁺ concentrations (20 mM) a Ca²⁺-Ca²⁺ exchange system was shown to operate, which is stimulated by Li⁺. The Na⁺-Ca²⁺ exchange system is capable of operating in the reverse direction, with Ca²⁺ uptake coupled with Na⁺ efflux. Such a vesicular preparation has the potential for providing useful experimental approaches to study the mechanism of this important Ca²⁺ extrusion system in the nervous system.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

Evidence that the cytosolic activity of 3-hydroxybutyrate dehydrogenase in chicken liver isL-3-hydroxyacid dehydrogenase

Classical fractionation studies showed that chicken liver contains two enzymes which can oxidize DL-3-hydroxybutyrate. The cytosolic enzyme is specific for the L-(+) isomer and accounts for 60% of the total activity. The mitochondrial activity is specific for the D-(?) isomer and accounts for 40% of the total activity. Kinetic studies showed that L-gulonic acid is a competitive inhibitor of the enzyme. We conclude that the cytosolic enzyme is the previously described L-3-hydroxyacid dehydrogenase.     

Structural state of the Na+ /d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na+-dependent phlorizin binding and Na+-dependent d-glucose transport

Target sizes of the renal sodium-d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at ?50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na⁺-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na⁺-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium-d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase a, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 μg/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID₅₀ of this venom inhibitor was about 2.5–5 μg/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca²⁺ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-238187. The [¹⁴C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E₁-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 μg/ml. Polylysine-induced platelet agglutination was not affected. β-Mercaptoethanol inactivated both its phospholiase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

Glucose inhibits 45Ca efflux from pancreatic β-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

Phosphatidylinositol hydrolysis and phosphatidylinositol 4′,5′-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca²⁺-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca²⁺ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of ³²PO₄ into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca²⁺-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This ³²PO₄ turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4′,5′-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca²⁺ deficiency. This phosphatidylinositol 4′,5′-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca²⁺ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4′,5′-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4′,5′-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca²⁺ mobilization.