Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

In vitro and in vivo inhibition of sucrose synthesis by sucrose

The inhibitory effects of sucrose on rates of sucrose synthesis by sucrose phosphate synthase (SPS) from the maize scutellum and on net rates of sucrose production in maize scutellum slices from added glucose or fructose were studied. Scutellum extracts were prepared by freezing and thawing scutellum slices in buffer. The extracts contained SPS and sucrose phosphate phosphatase, but were free of sucrose synthase. SPS activity was calculated from measurement of UDP formation in the presence of UDPG, fructose-6-P and sucrose. The ranges of metabolite concentrations used were those estimated to be in scutellum slices after incubation in water or fructose for periods up to 5 hr. UDPG and fructose-6-P also were added at concentrations that saturated SPS. At saturating substrate levels, sucrose inhibition of SPS was less than that when tissue levels of substrates were used. With tissue levels of substrates and sucrose concentrations up to ca 166 mM, sucrose inhibitions of sucrose synthesis in vitro by SPS were similar to those observed in vivo. However, as the sucrose concentration rose above 166 mM, SPS activity was not inhibited further, whereas there was a further sharp decline in sucrose production by the slices. It is concluded that sucrose synthesis in vivo is controlled by sucrose inhibition of SPS over a considerable range of internal sucrose concentrations.     

Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: Requirement for ADP

Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate (+ proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (K_m = 8.0 μm) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled α-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (>99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 μm), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 ± 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent K_I was 0.8 mm. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, α-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the mitochondrial NAD⁺-linked isocitric dehydrogenase is a major site for such control through the tricarboxylate cycle.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

A thin-layer chromatographic method for the quantitative separation and estimation of S-adenosylmethionine and S-adenosylethionine in rat liver

A procedure is described for the simultaneous isolation, separation, and quantitation of S-adenosylmethionine (AdoMet) and S-adenosylethionine (AdoEt) in rat liver. These compounds are isolated by precipitation with ammonium reineckate, are separated by thin-layer chromatography, and are quantitated by an isotope dilution determination. The procedure was tested with commercial standards added to liver homogenate ranging up to 160 μg AdoMet/g liver and 173 μg AdoEt/g liver; at all of the concentrations tested, the recoveries were linear and accurate. AdoMet recoveries were linear in the presence of 11.6 or 307 μg AdoEt/g liver, and AdoEt recoveries were linear in the presence of 37.8 or 191 μg AdoMet/g liver. AdoMet and AdoEt levels were measured in the livers of rats fed diet containing 0 or 0.3% dl-ethionine for 2 weeks. In the ethionine-treated animals, AdoMet concentrations were lower than in the controls; and, conversely, AdoEt increased from 0 to 259 μg/g liver.     

The action of 2-amino-4-(methylphosphinyl)-butanoic acid (phosphinothricin) and its 2-oxo-derivative on the metabolism of cyanobacteria and higher plants

When adequate concentrations of phosphinothricin (a potent inhibitor of glutamine synthetase) are added to Anacystis nidulans cells suspended in nitrate medium, ammonia excretion into the medium takes place. Similarly, when phosphinothricin is added to nitrogen fixing cultures of Anabaena ATCC 33047, ammonia is also released at high rates. Methionine sulphoximine, phosphinothricin and its 2-oxo-derivative (1 mM) stimulate ammonia production and cause a sharp drop in glutamine and asparagine concentrations, when fed to leaves of Triticum, Pisum and Helianthus. Less pronounced effects were detected with the leaves of a C₄ plant Zea.     

Inhibitors of T-cell mitogenesis: effect of mitogen dose.

The inhibitory effects of seven compounds (hydrocortisone, histamine, interferon, isoproterenol, norepinephrine, serotonin, and γ-aminobutyric acid) were tested in phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells as a function of PHA concentration. The cultures stimulated by suboptimal levels of PHA were much more sensitive to inhibition by the added compounds, with the curve of percentage inhibition versus concentration of inhibitor shifted to the left by greater than two orders of magnitude in all cases. At the lower doses of mitogen, concentrations of hydrocortisone, histamine, interferon, and isoproterenol which approach the concentrations of these or analogous compounds in vivo caused significant and substantial inhibition of the mitogen response.     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.     

Regulation of human in vitro anti-hapten responses: demonstration of a carrier effect

Human peripheral blood mononuclear cells, tonsillar lymphocytes, or mixtures of allogeneic or autologous B and T cells from these tissues were stimulated in vitro with the soluble hapten:carrier complexes TNP-OA, TNP-KLH, TNP-Myo, or TNP-Lac. These complexes were able to induce TNP-specific, direct PFC during 5–6 days in culture. The response involved proliferating PFC precursor B cells, nonproliferating T helper cells, and radiosensitive T-suppressor cell precursors. Exposure to high concentrations of free or carrier-bound hapten resulted in the inactivation of PFC precursor B cells. Carrier specific suppressor T cells could be induced by priming with nonhaptenated carrier protein and were able to block the PFC response when added to fresh target cultures. The use of hapten:carrier complexes permits the characterization of distinct T-cell functions independently of the assay system for B-cell activation.     

Fertilization in the sea urchin, Strongylocentrotus purpuratus is blocked by fluorescein dyes.

Fertilization in gametes of the sea urchin Strongylocentrotus purpuratus was reversibly inhibited by several analogs of the anionic dye fluorescein. The dyes acted very rapidly and were effective when added before or several seconds after insemination. Eggs and sperm did not appear to be irreversibly modified by incubation in seawater solutions containing tetraiodofluorescein (erythrosin B). Sperm binding to the vitelline layer was also inhibited by erythrosin B, but required concentrations greater than that necessary to block fertilization. The ability of the compounds to block fertilization was a function of the particular fluorescein derivative used and its concentration. The concentration required to inhibit fertilization in 50% of the eggs was related to dye lipid solubility. The dyes may inhibit fertilization by preventing gamete membrane fusion.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

Leaf production and nitrogen and phosphorus tissue content of Littorella uniflora (L.) Aschers. In relation to nitrogen and phosphorus enrichment of the sediment in oligotrophic Lake Hampen,Denmark

Littoral sediments with a dense population of Littorella uniflora (L.) Ascers. were artificially enriched with nitrogen and phosphorus. Responses to increasing levels of nutrients were recorded as leaf production and tissue nutrient content. Phosphorus enrichment resulted in increasing P concentrations in all plant fractions (leaves, stem and roots), whereas only leaves and roots showed increasing N concentrations when inorganic N was added. Leaf production increased significantly after increasing the sediment P level 2, 3 and 5 times. Nitrogen did not affect leaf production. The tissue content of total N and P in control plants averaged 3.29 and 0.28%, respectively. Thus the critical P level in L. uniflora seems to be about 0.28%, which is higher than previously published values. It was also found that L. uniflora translocates phosphorus from old to younger leaves.     

Disulfiram metabolism in isolated mesophyll cells and inhibition of photosynthesis and cyanide-resistant respiration

Tetraethylthiuram disulfide (disulfiram) stimulated medium acidification when added at a concentration of 0.4 millimolar to illuminated or nonilluminated suspensions of Asparagus sprengeri Regel mesophyll cells. Similar concentrations inhibited photosynthesis and cyanide-resistant respiration. The reduction product of disulfiram, diethyldithiocarbamic acid, accumulated in concentrations sufficient to account for the observed acidification.     

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions.

The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 mM Mn²⁺ and La³⁺ and, to a lesser extent, by 2–4 mM Ca²⁺ and Mg²⁺. Maturation induced by 1.5 mM Mn²⁺ was inhibited by K⁺ concentrations above 0.25 mM. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

Plots of turnover times versus added substrate concentrations provide only upper bounds to in situ substrate concentrations

In recent years it has become a common practice in the study of microbial activity in aquatic systems to estimate in situ substrate concentrations by plotting substrate turnover times (the substrate concentration divided by microbial uptake rate) against the concentrations of added radioactively labeled substrate. The rationale has been that the uptake rate will remain essentially constant if added concentrations of high specific activity substrates are small compared to in situ concentrations. Unfortunately, a correct mathematical analysis shows that the in situ concentrations estimated by this method are only upper bounds to the true in situ substrate concentrations, regardless of the size of the substrate spikes. The estimated in situ concentration will equal the true in situ concentration only if uptake rate is completely independent of substrate concentration, an unlikely situation in natural systems.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Effect of bacterial lipopolysaccharide on the in vitro secondary antibody response in mice. II. Abrogation of the suppressive capacity of endotoxin

Previous experiments have shown that bacterial endotoxin (ET) can be highly inhibitory to the in vitro secondary IgG antibody response when added 1–2 days after antigen. This paper examines the capacity of ET and another adjuvant, poly(AU), to circumvent the suppressive capacity of ET. It was found that ET or poly(AU) given simultaneously with antigen prevented any subsequent inhibition by ET added later to the cultures. Poly(AU) was effective in amounts as low as 1 μg/ml and ET in amounts as low as 0.1 μg/ml. Poly(AU) in greater amounts (50–100 μg/ml) also suppressed antibody synthesis when added 1–2 days after antigen, similar to ET. As with ET suppression, both ET and poly(AU) when added simultaneously with antigen were capable of overcoming the suppressive capacity of poly(AU). The capacity of small amounts of poly(AU) and ET to circumvent suppression in vitro by ET may help to explain why suppression by ET given after antigen has not been routinely observed in vivo. Lymphoid cells in vivo are most likely constantly exposed to either nuclear material released upon natural cell turnover or to ET from bacteria habitating the gut, resulting in an abrogation of any subsequent suppression by ET.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.