Hysteretic behavior of rat liver fructose 1,6-bisphosphatase induced by zinc ions

The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” K_m of about 2 μm. During the reaction the enzyme is converted in a slow process to a low K_m form (K_m is about 0.5 μm). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn²⁺ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate (O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem.249, 7228–7239) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account.     

Interaction of rabbit liver cathepsin M and fructose 1,6-bisphosphatase converting enzyme with their endogenous inhibitors

The stoichiometry of complex formation between two lysosomal proteinases from rabbit liver, cathepsin M and fructose 1,6-bisphosphatase converting enzyme (CE), and their respective endogenous inhibitors was studied by the equilibrium gel penetration method. In each case the molecular weight of the complex was found to be the sum of the molecular weights of the proteinase and its inhibitor, indicating the formation of 1:1 complexes. From the reappearance of proteinase activity on dilution, it is concluded that complex formation is reversible. Localization of the proteinase activities on the outer surface of the lysosomes was confirmed in these experiments by the inhibition of this proteinase activity on addition of inhibitors to intact lysosomes. The digestion by subtilisin of rabbit liver aldolase and rabbit liver fructose 1,6-bisphosphatase, the endogenous substrates for the lysosomal proteinases, was unaffected by the inhibitors.     

A one-step procedure for the isolation of fructose 1,6-bisphosphatase and fructose 1,6-bisphosphate aldolase from rabbit liver

Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 18,650}$, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{? 135,00}$). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the $\text{A}{}_{610}\text{A}{}_{280}$ ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH₂-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.     

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.     

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.     

Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.     

Location of lysyl residues at the allosteric site of fructose 1,6-bisphosphatase

Various flavin analogs were used as alternate substrates or competitive inhibitors to characterize the FMN binding sites of the NADH- and NADPH-specific FMN oxidoreductases from Beneckea harveyi. Several polyhydroxyl compounds were found to be poor competitive inhibitors for the FMN sites of these enzymes. The FMN binding sites of the two enzymes were found to be quite similar. The NADH:FMN oxidoreductase binds FMN exclusively through the isoalloxazine ring. The methyl groups at positions 7 and 8 contribute significantly to this binding. Utilizing lumichrome as a competitive inhibitor of the FMN binding site and AMP as a competitive inhibitor of the NADH binding site, we were able to determine that the NADH:FMN oxidoreductase forms an active ternary complex with NADH binding first in an ordered mechanism. The NADPH oxidoreductase also binds FMN primarily through the isoalloxazine ring. Unlike their participation in reaction with the NADH-specfic enzyme, the methyl groups at positions 7 and 8 are not involved in binding. There was no significant binding of the ribityl phosphate moiety with either enzyme. Both enzymes have lower K_m values for lumiflavin than FMN.     

Rabbit liver fructose-1,6-bisphosphatase: location of an active site lysyl residue in the COOH-terminal fragment generated by a lysosomal proteinase

Digestion of native rabbit liver fructose-1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) with a membrane-bound proteinase from rat liver lysosomes yields a fragment of M_r = 9850. This peptide contains the COOH terminus of the Fru-P₂ase polypeptide chain and also the cyanogen bromide peptide (BrCN5) carrying the active site lysyl residue. The sequence of BrCN5 and its location with respect to the COOH terminus of the polypeptide chain have been determined. The active site lysyl residue is located at approximately residue ?54 from the COOH terminus. The bond hydrolyzed by the lysosomal proteinase is located between residues ?88 and ?89 from the COOH terminus.     

Induction of fructose 1,6-bisphosphatase and glucose 6-phosphatase in fetal mouse liver

Fructose 1,6-bisphosphatase and glucose 6-phosphatase were induced in organ cultures of liver tissues from 15- and 19-day-old fetal mice, using a culture method that allowed the tissues to be maintained for 7 days in the absence of serum. In cultures from 15-day-old fetal liver, both enzyme activities increased significantly per milligram of DNA after a lag period of 1 to 3 days. In cultures from 19-day-old fetal liver only glucose 6-phosphatase increased in the absence of inducer. N6,O2'-Dibutyryladenosine 3',5'-monophosphate enhanced the rate of increase in fructose 1,6-bisphosphatase and glucose 6-phosphatase activities. The minimum effective concentration of the cyclic nucleotide was approximately 10(-6) M. Dexamethazone inhibited the increase in fructose 1,6-bisphosphatase during culture for 7 days. Glucose 6-phosphatase activity was enhanced by dexamethazone in cultures from 19-day-old fetal liver, but was without effect on glucose 6-phosphatase in cultures from 15-day-old fetal liver. The minimum inhibitory concentration of dexamethazone was less than 10(-8) M. The results suggest a complicated effect of the cyclic nucleotide on the two enzymes in fetal mouse liver as well as different mechanisms of action of dexamethazone on the induction of two enzymes.     

Role of ATP in the cleavage mechanism of the EcoP15 restriction endonuclease

The EcoP15 restriction endonuclease forms complexes at specific sites on unmodified DNA both in the presence and in the absence of S-adenosyl-l-methionine. ATP acts as an allosteric effector of EcoP15 and induces DNA cleavage followed by release of the enzyme from the DNA. The efficiency of endonucleolytic scission varies from site to site. The nucleotide sequences at sites that are cleaved at a high frequency were compared.     

Activation volumes for the two steps of the conjugate-base mechanism in liquid ammonia

The pressure dependence (10–4000 bar) of the kinetics of the ammoniation of[Co(NH₃)₅X](ClO₄)₂ (X = N₃, Cl) and the isomerization of [Co(NH₃)₅(ONO)](ClO₄)₂ in liquid ammonia is reported. The conjugate-base mechanism is operative for these complexes over the entire pressure range used. Activation and thermodynamic parameters were obtained for each of the two steps of the mechanism for [Co(NH₃)₅(N₃)](ClO₄)₂ at 20 bar. Values for the overall activation volume extrapolated to zero pressure are ΔV³(0) = ?12 (11.35 °C, ONO); ?20 (24.45 °C, N₃) and ?30 (0.50 °C, Cl) cm³ mol^?1. Application of El'yanov and Hamann's empirical relation for the pressure dependence of the ionization of weak acids separates the contributions of the pre-equilibrium (ΔV_CB⁰) and the elimination or isomerization reaction (ΔV₂³) (at zero pressure). The values obtained for [Co(NH₃)₅X](ClO₄)₂ are (givens as X; ΔV_CB⁰ and ΔV₂³ in cm³ mol^?1; T in °C): (ONO; ?16 and ?15; 11.35), (N₃; ?22 and 1;24.45), (Cl; ?22 and 3;0.50). These values fit in the accepted picture of volume effects in cobalt(III) ammine kinetics.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

On the use of trace levels of [1-14C]galactose to estimate cycling between fructose 6-phosphate and fructose diphosphate

The [1-¹⁴C]galactose method for estimating phosphofructokinase flux in liver preparations (R. Rognstad and J. Katz, 1976, Arch. Biochem. Biophys.177, 337–345) has been evaluated using a computer program that calculates the flow of ¹⁴C label through all the carbon atoms of all the intermediates in a metabolic system. Our computations show that this method is subject to relatively small errors when gluconeogenesis from dihydroxyacetone proceeds in the absence of glycogenolysis or of phosphorylation of exogenous glucose, provided that bidirectional flux through aldolase is at least twice the rate of gluconeogenesis. Significant error may be introduced into estimates of phosphofructokinase flux if the experimental system deviates from these conditions.     

Positive surface charge inhibition of phospholipase A2 in mixed monolayer systems

Inhibition of pancreatic phospholipase A₂ by surface-active local anesthetics was recently reported by this laboratory to be due to enzyme-anesthetic interaction in the subphase and surface effects. In order to study surface effects in the absence of subphase effects, a long-chain tetracaine analog which was completely insoluble in the subphase, dimethylaminoethyl p-decoxybenzoate, was synthesized. To determine if inhibition was due to the positive surface charge of the analog or some other effect related to structure, the analog's inhibitory effects were compared with those of octadecylamine. Analog-didecanoyl lecithin (PC) monolayers showed nonideal mixing as evidenced by a condensing effect, while octadecylamine-didecanoyl PC monolayers showed ideal mixing. The apparent pK′_a of octadecylamine-dioctanoyl PC micelles (1:4) was 9.9, while that of the analog-dioctanoyl PC micelles (1:4) was 7.6. At pH values where both amines were fully protonated, inhibition of both porcine pancreatic and Crotalus adamanteus phospholipase A₂ on the mixed films was maximal and similar (94–97%). Inhibition decreased with increasing pH and decreasing surface charge on both mixed films and at pH values where both amines were 50% protonated, inhibition was half-maximal. At pH 8.5, where the analog was unprotonated, no inhibition was observed. Thus, inhibition of phospholipase A₂ appears to be due to a positive surface charge alone rather than any effects related to anesthetic structure or spacing in the monolayer.     

Oxygen-18 studies on the oxidative deamination mechanism of alicyclic primary amines in rabbit liver microsomes

The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of ¹⁸O₂ or in a medium containing H₂¹⁸O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.5–336}\text{min}$) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium, Chromatium vinosum. III. Absence of extrachromosomal DNA

Inducible formation of ribulose-1,5-bisphosphate (RuBP) carboxylase in the cells of Chromatium vinosum under autotrophic conditions was not affected by six different inhibitors of DNA synthesis. Photosynthetic CO2 fixation and RuBP carboxylase activities were not influenced by seven reagents known to eliminate plasmids. Plasmids were not detectable by agarose gel electrophoresis employing either the cleared lysate or alkaline sodium dodecyl sulfate method, nor were they detected by ethidium bromide-CsCl density gradient centrifugation. Overall experimental results tend to indicate that plasmids are absent in the Chromatium cells and that the induction of RuBP carboxylase is presumably not regulated in the DNA replication process.     

The mechanism of anion inhibition of pork heart aspartate aminotransferase

The mechanism of anion inhibition of the reaction of the pork heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1) with erythro-β-hydroxy-l-aspartate was investigated. This reaction produces a mixture of complexes, one of which is characterized by an absorption maximum at 492 nm. Spectrophotometric analysis of equilibrium mixtures of aspartate aminotransferase and erythro-β-hydroxy-l-aspartate, in different buffers, indicated that acetate, chloride, and cacodylate were competitive inhibitors of hydroxyaspartate binding. Pyrophosphate, however, was not a competitive inhibitor. Between pH 4.5 and 9.0 the affinity of the enzyme for the monovalent anions decreased as the pH increased. The data indicated that the anion binding group had a pK_a in the range from pH 6 to 7, depending upon the anion studied. From pH 4.5 to 9.0, the substrate dissociation constant and the distribution of enzyme-substrate complexes were both unaffected by pH. By stopped-flow spectrophotometry, an initial rapid relaxation ($\text{t}\text{1}\text{2}\text{= 2–8}\text{ms}$) was associated with an increase in absorbance at 492 nm, and this rate depended upon both substrate and buffer concentrations. A slower relaxation ($\text{t}\text{1}\text{2}\text{= 180}\text{ms}$) was associated with a decrease in the absorbance at 492 nm to approximately 70% of the value attained in the first rapid reaction. The rate of this slower reaction was largely independent of substrate and buffer concentrations. Kinetic analysis of the rates of the first relaxation in several different concentrations of Tris-acetate buffer of pH 8 showed that the rate of association decreased with increasing acetate concentration whereas the reaction rate for dissociation was unaffected. Thus, acetate appears to exert its inhibitory effect by preventing the formation of the enzyme-substrate complex rather than by displacing the substrate from the enzyme.     

Genes and pseudogenes for human U2 RNA: Implications for the mechanism of pseudogene formation

Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3′ end. The conserved 3′-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus.The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5′ and 3′ sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5′ side the homology continues beyond the Alu repeats whereas on the 3′ side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.     

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10^?4m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC^? lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC^? enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC^? adenylate cyclase utilized MgATP^2? as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC^? enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP^2?. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.