The role of cyclic AMP in neoplastic cell growth and regression. II. Growth arrest and glucose-6-phosphate dehydrogenase isozyme shift by dibutyryl cyclic AMP

N⁶,O²′-Dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) injected into rats bearing MTW9 mammary carcinoma resulted in an early disappearance of tumor microsomal glucose-6-phosphate dehydrogenase activity while mitochondrial and supernatant isozyme activities were not affected. Prolonged DBcAMP treatment of rats bearing 5123 hepatoma significantly decreased all glucose-6-phosphate dehydrogenase isozyme activities but did not alter host liver isozyme activities or liver regeneration. Since DBcAMP treatment arrested growth of these tumors, the loss of microsomal glucose-6-phosphate dehydrogenase may be an early event in the inhibition of tumor growth $\text{in vivo}$.     

Mouse spermatozoal glucose-6-phosphate dehydrogenase is the X-linked form

Mouse spermatozoal “glucose-6-phosphate dehydrogenase” was characterized for substrate utilization, electrophoretic mobility, and by immunoinactivation with an antibody to human, erythrocytic glucose-6-phosphate dehydrogenase. The enzymatic activity was found to have the properties of the X-linked form ($\text{Gpd-2}$).     

Immunological titration of rat liver glucose-6-phosphate dehydrogenase from animals fed high and low carbohydrate diets.

Recent work (Hizi and Yagil [1974] Eur. J. Biochem. $\text{45}$: 211–221, and Kelly $\text{et. al.}$ [1975] Fed. Proc. $\text{34}$: 881) suggests that the marked increase in rat liver glucose-6-phosphate dehydrogenase activity which is observed upon feeding an animal a high carbohydrate diet does not involve an increase in the total amount of enzyme present. In contrast, the data presented herein involving immunological titrations of rat liver glucose-6-phosphate dehydrogenase indicates that the increase in enzyme activity resulting from feeding a high carbohydrate diet does involve an increase in the total amount of enzyme present.     

Bakers' yeast uridine nucleosidase is a regulatory copper containing protein.

The enzymatic properties of homogeneous bakers' yeast uridine nucleosidase, prepared as previously described (G. Magni et al., J. Biol. Chem. 1975 $\text{250}$, 9–13) have been further investigated, and in addition to glucose-6-phosphate and ribose the enzyme activity was inhibited by ribose-5-phosphate and ribulose-5-phosphate. The curves describing this inhibitions were sigmoidal and when the data were plotted according to Hill, n′ values different from 1 were observed suggesting the existence of interactions among the inhibitory molecules binding sites. Furthermore the percentage of inhibition exerted by glucose-6-phosphate, ribose and ribose-5-phosphate on the enzyme activity varied at different pH values. The addition of various chelating agents to the activity assay mixture caused a strong inhibition of the enzyme activity and metal analysis by atomic absorption spectrophotometry, colorimetric methods and electronic paramagnetic resonance, indicated the presence of 1 copper atom per enzyme molecule. Finally the inhibition exerted by metal ions on the enzyme activity was described.     

An electron cytochemical study of 6-phosphogluconate dehydrogenase activity in infected erythrocytes during malaria

The distribution of 6-phosphogluconate dehydrogenase (6-PGD) was examined by an electron microscopic technique in erythrocytes of rodents infected with $\text{Plasmodium berghei}$, chickens with $\text{P. gallinaceum}$ and rhesus monkeys with $\text{P. knowlesi}$. Unlike glucose-6-phosphate dehydrogenase, which is restricted to the host erythrocyte, 6-PGD was found to be present in the parasite as well as the host erythrocyte in all infections studied. The implications of these findings are discussed in relation to the metabolism of malaria parasites.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes

In view of the possible utilization of aphidicolin, a specific inhibitor of DNA polymerase α, in the treatment of neoplastic diseases, it seemed important to assess the mutagenic effect of the drug and the possible modification induced by metabolic activation in the liver. This paper shows that aphidicolin lacks mutagenicity in the Ames' $\text{Salmonella}$-microsome test in agreement with our previous observation that it does not induce DNA repair synthesis in HeLa cells. During the studies of mutagenicity we have observed that aphidicolin is converted to inactive derivative(s) by rat liver microsomal oxidases. The reaction is dependent on time and temperature and requires NADP⁺ and glucose-6-P. The metabolites are not mutagenic and they do not induce DNA repair synthesis in HeLa cells. Therefore the possible anti-cancer use of aphidicolin is not hampered by its partial metabolic inactivation in liver. Our results suggest however that aphidicolin will possibly be clinically useful at concentrations higher than those expected from our studies with human DNA polymerase $\text{α}\text{in vitro}$ and human neoplastic cell lines $\text{in vivo}$. The metabolic derivative(s) of aphidicolin is inactive both against cellular DNA polymerase α and Herpes simplex viral DNA polymerase.     

Quantitative requirements for NADPH in the support of aromatization by human placental microsomes

Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes. 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity. Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system. With excess $\text{Leuconostoc mesenteroides}$ glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 μM was observed for aromatization which is significently below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome $\text{c}$ reductase. These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.     

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of $\text{N-}\text{ethylmaleimide}$. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘$\text{W/S}$’ ratio. For latent microsomes, the value of this parameter was determined to be $\text{0.65 ± 0.02}$ and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The $\text{W/S}$ ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The $\text{W/S}$ ratio was observed to be dependent upon the method of microsome preparation yielding values of $\text{1.02 ± 0.02}$ for ‘hypertonically disrupted’ vesicles and $\text{1.28 ± 0.02}$ for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome $\text{P-450}$ and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation.     

Evidence that ATP exerts control of the rate of glucose utilization in the intact Escherichia coli cell by altering the cellular level of glucose-6-P, an intermediate known to inhibit glucose transport in vitro

The effects of treating nitrogen-starved cultures of $\text{Escherichia coli}$ W4597 (K) with various doses of 2,4-dinitrophenol include increases in the rates of glucose utilization, decreases in ATP and glucose-6-P and maintenance of the level of fructose-1, 6-P₂. A quantitative correlation was observed between the increases in the rates of glucose utilization and decreases in glucose-6-P in agreement with the observation made $\text{in vitro}$ that glucose-6-P inhibits glucose transport in $\text{E. coli}$. A quantitative correlation was also observed between glucose-6-P and ATP indicating that the fall in glucose-6-P is effected by the fall in ATP which indirectly signals increased glucose utilization and increased ATP production.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

The stereospecificity of D-glucose-6-phosphate: 1L-myo-inositol-1-phosphate cycloaldolase on the hydrogen atoms at C-6

D-Glucose-6-phosphate: 1L-$\text{myo}$-Inositol-1-phosphate cycloaldolase from rat testis or mammary gland removed stereospecifically the pro-S hydrogen atom at C-6 from D-glucose-6-phosphate. The pro-R hydrogen at C-6 remained in the product, 1L-$\text{myo}$-Inositol-1-phosphate and evidence is given that it is the hydrogen at C-1 of 1L-$\text{myo}$-Inositol-1-phosphate. The possible mechanism of cyclization is discussed.     

Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila.

A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain $\text{DBA}\text{2J}$) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.     

Inactivation and reactivation of hexokinase type II

Hexokinase isozyme II which loses activity rapidly in the absence of glucose ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$) is stabilized in the presence of glucose-6-P, P_i and ADP when glucose is also present but not by kinetically inert analogs. Enzyme inactivated by incubation in the absence of glucose is fully and rapidly recovered ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>～-\; 10\; </mtext><mtext>min</mtext>}}$) by addition of both glucose and mercaptoethanol, each at 0.1 m. In the presence of 0.1 mm glucose, both glucose-6-P and P, facilitate the reactivation. Reactivation proceeds in two steps both with unfavorable equilibria: a fast reduction followed by a slow renaturation. Native enzyme is much more resistant to irreversible inactivation by trypsin than is enzyme that has lost its activity by incubation in the absence of glucose. The latter form shows no protection from trypsin action by glucose. Streptozotocin-diabetic rats that have lost hexokinase II preferentially in their insulin-sensitive tissues do not contain an activatable form of hexokinase in at least one of these, heart. The greater sensitivity of inactivated hexokinase to denaturation by trypsin suggests that such a “reservoir” form may be destroyed rapidly in vivo. Glucose may be important in determining the steady-state level of hexokinase II by “guiding” the folding of translation product. In this view insulin would act through its effect on glucose permeability.     

Measurement of phosphate esters in extract of fetal rat heart by automated high pressure liquid chromatography

Creatine phosphate, nucleotides and glycolytic phosphate esters were estimated in extract of beating, in situ freeze clamped, $\text{131}\text{2}$ to $\text{191}\text{2}$ day fetal rat hearts by automated phosphate ester chromatography. Creatine phosphate increased more than 4-fold to almost 9 n moles per mg. protein at $\text{191}\text{2}$ days, while ATP remained relatively constant at about 19 to 21 n moles per mg. protein. Most other nucleotides decreased as gestation advanced. ATP rather than creatine phosphate appears to be the major energy source of fetal rat heart. Except for glucose-6-phosphate, which increased, the glycolytic phosphate esters decreased only very slightly with advancing gestational age, suggesting a relatively stable basal glycolytic activity. Methodology includes correction for phosphate esters of whole blood trapped in extracts of in situ freeze clamped tissues.     

Evidence for an allosteric control of the pentose phosphate pathway in Candida utilis

Glucose 6-phosphate dehydrogenase from $\text{Candida utilis}$ exists in two interconvertible species having molecular weights of 110,000 and 220,000, respectively. By use of sodium dodecyl-sulfate the enzyme can be dissociated into 2 electrophoretically separable subunits. The native enzyme was shown to be strongly inhibited by low concentrations of erythrose 4-phosphate and glyceraldehyde 3-phosphate, respectively. Since both these sugar phosphates are metabolites in the later course of the oxidative pathway this regulation may be considered as a negative feedback control of the pentose phosphate cycle.     

An invitro characterization of interstrand cross-links in DNA exposed to the antitumor drug cis-dichlorodiammineplatinum(II)

The $\text{cis}$-isomer of the antitumor drug dichlorodiammineplatinum(II) [$\text{cis}$-Pt(II)] was tested for its abilty to introduce nicks (single-strand breaks) into supercoiled PM2 DNA. Whereas incubations up to 24 h show no indication of $\text{cis}$-Pt(II)-treated DNA having single-strand breaks, DNA interstrand cross-links were detected in the first 15 min of incubation. Furthermore, the formation of DNA interstrand cross-links was $\text{both}$ inhibited and fully reversed after incubation with 2 mM thiourea.     

Alpha-1-acetylmethadol and its N-demethylated metabolites have potent opiate action in the guinea pig isolated ileum

α-1-Acetylmethadol (LAM) and its N-demethylated metabolites α-1-noracetylmethadol (NAM) and α-1- dinoracetylmethadol (NNAM) exhibited opiate-like actions $\text{in}$$\text{vitro}$ by depressing the electrically induced twitch of the longitudinal muscle of the guinea pig ileum. These effects were reversed by the opiate antagonist naloxone. The effect of both NAM and NNAM was approximately 15 times greater than LAM. The 50 percent inhibitory concentrations of the metabolites were well below those levels in plasma reported for man. The long duration of action of LAM as an opiate substitute is most likely due to the conversion to its metabolites.     

Enzymic properties of phosphonic analogues of D-erythrose 4-phosphate

4-Deoxy-D-$\text{erythro}$ tetrose 4-phosphonate and 4,5 dideoxy D-$\text{erythro}$ pentose 5-phosphonate, the phosphonic analogues of D-erythrose 4-phosphate, have been prepared by oxidation of the corresponding analogues of glucose 6-phosphate and tested as substrates of 3-deoxy-D-$\text{arabino}$ heptulosonate 7-phosphate synthetase, transaldolase and transketolase. Kinetic parameters of the reaction with the phosphonate analogues and the natural substrate have been compared.     

In vitro stimulation by 2,4-dichlorophenoxyacetic acid of an ATPase and inhibition of phosphatidate phosphatase of plant membranes.

The auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was shown to modulate the activities of several phosphatases with membranes isolated from soybean hypocotyls under conditions where degradative changes in the membranes were minimized. The medium for isolation of membranes consisted of 0.1 M Tris/HCl or Tris/acetate, pH 6.5, 0.5 M sucrose, 4% choline ($\text{w}\text{w}$) and 4% ethanolamine ($\text{v}\text{v}$) to inhibit phospholipase D, 20 mM EGTA [ethyleneglycol-bis- (β aminoethyl ether) N,N-tetracetic acid] and 1 mM nupercaine, to inhibit phospholipase A. In contrast, the inactive auxin analog 2,3-D, did not influence ATPase activity. Endogenous release of inorganic phosphate from an unidentified source was also stimulated 30% by 2,4-D. Phosphatidate phosphatase was inhibited by 2,4-D, whereas hydrolysis of glucose-6-phosphate was not influenced by 2,4-D under the same conditions. These observations may be of relevance to the proton pump hypothesis of growth regulation.