Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid, Loligo pealeii. Role of the enzyme in energy production from amino acids.

1. The activity of glutamate dehydrogenase was measured in the tissues of the squid, Loligo pealeii. The enzyme occurs in high activity in digestive pouch, systemic heart, and all muscle tissues. 2. Glutamate dehydrogenase from mantle muscle is located intra-mitochondrially, has a molecular weight of 310,000, and is electrophoretically similar to the enzyme from all other squid tissues. 3. The enzyme from mantle muscle was purified 40-fold by elution from DEAE-cellulose and used for kinetic studies. The enzyme is NAD+-specific, activated by ADP, AMP, and leucine, and inhibited by GTP, GDP, ATP, and reaction products (in particular NADH). 4. Squid glutamate dehydrogenase shows an almost absolute dependence on ADP. The purified enzyme is activated over 100-fold by saturating concentrations of ADP (Ka = 0,75 7M); The pH optima are also altered significantly by ADP. 5. The enzyme appears to be kinetically adapted to favour glutamate oxidation in comparison to glutamate dehydrogenase from other resources. The evidence indicates that the primary role of glutamate dehydrogenase in squid mantle muscle is in regulating the catabolism of amino acids for energy production.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli. 2. Evidence for two different active sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase by substrate analogues, by the end product, tyrosine, and by the protein modifying agent iodoacetate has been investigated. The purpose of the investigations was to determine if the two reactions catalyzed by the enzyme occur at a single active site or at two separate active sites. Evidence in support of the conclusion that the mutase and dehydrogenase reactions are catalyzed at two similar but distinct active sites comes from the following results: (1) A substrate analogue (endo-oxabicyclic diacid) that inhibits competitively the mutase reaction has no effect on the dehydrogenase reaction. (2) Malonic acid and several of its derivatives act as inhibitory analogues of chorismate in the mutase reaction and of prephenate in the dehydrogenase reaction. However, different dissociation constants for their interaction with the free enzyme are obtained from studies on the mutase and dehydrogenase reactions. (3) The kinetics of the inhibition by tyrosine of the mutase reaction in the presence of NAD differ from those of the dehydrogenase reaction. The results confirm that carboxymethylation with iodoacetate of one cysteine residue per subunit eliminates both mutase and dehydrogenase activities and show that the inactivation of the enzyme activities is due to iodoacetate functioning as an active site directed inhibitor.     

Repeating functional domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Each polypeptide chain in the lipoate acetyltransferase (E2) core of the pyruvate dehydrogenase complex from Escherichia coli contains three repeating sequences in the N-terminal half of the molecule. The repeats are highly homologous in primary structure and each includes a lysine residue that is a potential site for lipoylation. We have shown that all three sites are lipoylated, at least in part, and that the three lipoylated segments of the E2 chain can be isolated as distinct functional domains after limited proteolysis. Each domain becomes partly acetylated in the intact complex in the presence of substrate. In the primary structure, the domains are separated by regions of polypeptide chain oddly rich in alanine and proline residues. These regions are probably the conformationally mobile segments observed in the 1H-n.m.r. spectrum of the complex and which are removed by tryptic cleavage at Lys-316. The C-terminal half of the molecule contains the acetyltransferase active site and the binding sites for E1, E3 and other E2 subunits. The pyruvate dehydrogenase complex of E. coli, which has a heterogeneous quaternary structure, is thus far unique among the 2-oxo acid dehydrogenase complexes in possessing more than one lipoyl domain per E2 chain, but this may be a general feature of the enzyme from Gram-negative organisms.     

Phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in isolated adipocytes. Effects of 2-oxo acids.

Isolated adipocytes from rat epididymal fat-pads were incubated with [32P]Pi, and intracellular phosphoproteins were then analysed by SDS/polyacrylamide-gel electrophoresis and autoradiography. A phosphorylated polypeptide of apparent Mr 46,000 was identified as the alpha-subunit of branched-chain 2-oxo acid dehydrogenase complex by immunoprecipitation using antiserum raised against the homogeneous E1 component of branched-chain 2-oxo acid dehydrogenase complex. Immunoprecipitation of this phosphoprotein is blocked in a competitive manner by purified branched-chain 2-oxo acid dehydrogenase complex. Peptide mapping of the isolated phosphoprotein indicates that two sites on the polypeptide are phosphorylated in the intact cells. Addition of branched-chain 2-oxo acids to the incubation medium causes diminution in the extent of labelling of both phosphorylation sites on the alpha-subunit, an effect presumably mediated via their known inhibitory action on branched-chain 2-oxo acid dehydrogenase kinase. These observations provide direct evidence for phosphorylation of branched-chain 2-oxo acid dehydrogenase complex in intact cells.     

Multiplicity of phosphorylation sites onEscherichia coli isocitrate dehydrogenase

Several lines of evidence indicate that the in vivo phosphorylation of isocitrate dehydrogenase (EC 1.1.1.42) inEscherichia coli occurs at multiple sites: first, the phosphorylated enzyme can be resolved by two-dimensional electrophoresis into three distinct spots differing in charge; second, the analysis of its phosphoamino acid content shows that it is modified at both serine and threonine residues; third, its extensive hydrolysis by proteolytic enzymes yields several different phosphopeptides.     

Meiosis in allopolyploid Crepis capillaris. II. Autotetraploids.

Meiotic chromosome pairing of autotetraploid Crepis capillaris was analysed by electron microscopy of surface-spread prophase I nuclei and compared with light microscopic observations of metaphase I chromosome configurations. Prophase I quadrivalent frequencies are high in all three tetrasomes. (A, D, and C) and partially dependent on chromosome size. At metaphase I quadrivalent frequencies are much lower and strongly dependent on chromosome size. There is no evidence for multivalent elimination during prophase I in this system, and the reduction in multivalent frequency at metaphase I can be explained by an insufficiency of appropriately placed chiasmata. The high frequencies of prophase I quadrivalents far exceed the two-thirds expected on a simple model with two terminal independent pairing initiation sites per tetrasome, suggesting that multiple pairing initiation occurs. Direct observations reveal relatively high frequencies of pairing partner switches (PPSs) at prophase I, which confirms this suggestion. The numbers of PPSs per tetrasome show a good fit to the Poisson distribution, and their positional distribution along chromosomes is random and nonlocalized. These observations favour a model of pairing initiation based on a large number of evenly distributed autonomous pairing sites each with a uniform and low probability of generating a PPS.     

Fluorescence studies of threonine-promoted conformational transitions in aspartokinase I using the substrate analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate

The trinitrophenyl derivative of ATP, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, has been used as a spectroscopic probe to investigate threonine-promoted conformational changes in the aspartokinase region of aspartokinase-homoserine dehydrogenase I in an attempt to relate the structural effects of threonine binding to inhibition of enzymatic activity. Binding of this analogue substrate to the enzyme is characterized by a 9-fold enhancement in probe fluorescence. Saturating levels of the feedback inhibitor, threonine, produce a 77% increase in fluorescence enhancement, indicating an increase in the rigidity or hydrophobicity of the nucleotide-binding site in the inhibited form of the enzyme. Threonine titration studies indicate that the two inhibitor-binding sites found on each subunit do not contribute equally to the fluorescence-detected conformational change. Comparison of the spectral change with the inhibition of dehydrogenase activity has revealed the exclusive involvement of the non-kinase threonine sites. No transition can be detected as a consequence of inhibitor binding at the kinase subsites. The results of the 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate study have provided further evidence for a concerted kinase-dehydrogenase conformational change which is induced by threonine interaction with the high affinity binding sites and which provides maximal inhibition of homoserine dehydrogenase and the majority of aspartokinase inhibition. The failure to observe a distinct enzyme form produced by threonine occupation of the low affinity kinase sites suggests that no large structural reorganization of the kinase active site is produced as a result of this binding event. The conformational change, suggested by the cooperativity of threonine binding, must instead involve only a subtle or highly localized alteration which does not perturb the environment of the ATP-binding cleft.     

Chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes: evidence that the two reactions occur at one active site.

The relationship between the sites for catalysis of two reactions by the bifunctional enzyme chorismate mutase--prephenate dehydrogenase has been investigated. The results are consistent with the occurrence of both reactions at one active site. Comparisons have been made between experimental data for the time course of the overall reaction and computer simulations, according to various models for the relationship between the mutase and dehydrogenase sites. A model based on a single active site is consistent with the time course data if a minor proportion of the chorismate that reacts can be converted through to (hydroxyphenyl)pyruvate without the intermediate release of prephenate. Consistent with this requirement, some channeling of radioactivity from chorismate to (hydroxyphenyl)pyruvate has been detected. A model based on two separate sites has also been considered; the simulations show that if this model applies there is no need to postulate any channeling of the intermediate, prephenate, between the sites and there must be marked inhibition of the dehydrogenase reaction by chorismate. Since channeling has been observed and chorismate increases the dehydrogenase rate under all conditions, the two-site model appears unlikely. Consistent with the one-site model are the observations that a variety of inactivating conditions cause parallel loss of mutase and dehydrogenase activity and that identical protection against inactivation of both mutase and dehydrogenase by iodoacetamide is afforded by prephenate.     

Origins of the mycoplasmas: sterol-nonrequiring mycoplasmas evolved from streptococci.

We report the establishment of a phylogenetic relationship between the sterol-nonrequiring mycoplasmas (Acholeplasma species) and streptococci. Three specific antisera prepared against purified Streptococcus faecalis fructose diphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and Pediococcus cerevisiae glyceraldehyde-3-phosphate dehydrogenase were used for comparative enzyme immunological studies; the Ouchterlony double-diffusion technique and the quantitative microcomplement fixation procedure were employed. The reactions obtained provide evidence showing that all seven ACholeplasma species studied (A. laidlawii, A. granularum, A. modicum, A. oculi, A. axanthum. A. hippikon, and A. equifetale) are phylogenetically related to streptococci and that they evolved from streptococci. The data strongly suggest that the acholeplasmas comprise a distinct evolutionary group that has diverged from streptococci belonging to Lancefield group D or N. No reactions were observed between these enzyme antisera and cell extracts from six fermentative Mycoplasma species. These results support the view that mycoplasmas are derived from various bacteria.     

Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes

This report presents evidence for diversity in membrane binding sites between three forms of ankyrin: brain ankyrin, erythrocyte ankyrin, and a variant of erythrocyte ankyrin (protein 2.2) present in circulating human erythrocytes that is missing a regulatory domain. These ankyrins were compared with respect to binding to kidney microsomes and exhibited the following behavior. 1) Brain and erythrocyte ankyrin each bind to distinct sites. 2) Protein 2.2 is an activated ankyrin that binds to all of the sites accessible to both brain and erythrocyte ankyrin and, in addition, associates with its own specialized sites. 3) The specificity of these membrane sites for various ankyrins is not absolute but reflects 2.5-10-fold differences in relative affinities. Further evidence that binding sites of different ankyrins share some common features is that the cytoplasmic domain of the erythrocyte anion transporter associates with all three ankyrins and displaces binding of the ankyrin variants to kidney membranes. The differences between erythrocyte and brain ankyrins in association with kidney membranes are likely to have physiological relevance to kidney because immunologically related isoforms of ankyrin are expressed in this tissue: erythroid ankyrin which is restricted to the basolateral domains of two cell types and a brain-related ankyrin expressed in all cells and present on apical as well as basolateral membrane surfaces. An unanticipated observation was the discovery of a membrane-associated ankyrin protease in kidney that is specific for erythrocyte ankyrin and may selectively activate the erythroid isoform of ankyrin. The variety of binding sites within this group of ankyrin proteins supports the idea that ankyrins are capable of linking a number of different membrane proteins to the spectrin-actin skeleton.     

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation.

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.     

Differences in the interactions of liver alcohol dehydrogenases with probes binding into the substrate pocket

The interactions of three groups of probes (berberine alkaloids, tricyclic psychopharmaca and acridine derivatives) with isoenzymes of horse liver alcohol dehydrogenase and with rat liver alcohol dehydrogenase have been examined. These compounds inhibit the activity of the EE isoenzyme of horse liver alcohol dehydrogenase but differ in their behaviour towards the steroid-active enzymes (i.e. the ES isoenzyme of horse liver alcohol dehydrognase and alcohol dehydrogenase from rat liver): psychopharmaca inhibit, acridines activate and berberines do not bind. The ligands differ also in their influence on the modification of the EE isoenzyme by iodoacetate. Polarities (expressed as Kosower's Z values) of the respective binding sites on the EE isoenzyme were estimated from optical properties of bound probes. Berberines bind into a very hydrophobic area of the enzyme molecule, the binding site for psychopharmaca is moderately hydrophobic and that for acridines is rather polar. Steric arrangements of the binding sites are also discussed. The data presented confirm the existence of three distinct binding sites for these ligands in the substrate pocket of liver alcohol dehydrogenase.     

Inactivation of E. coli pyruvate formate-lyase: role of AdhE and small molecules

Escherichia coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectable, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both beta-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.     

Hexaamminecobalt(III) binding environments on double-helical DNA.

Previous cation nmr evidence suggests that univalent cations such as Na+ bind to DNA in a diffuse, nonspecific manner, whereas di- and trivalent cations show distinct binding heterogeneity. Here are reported 59Co- and 23Na-nmr measurements of the %GC dependence of the DNA binding behavior of the trivalent hexaamminecobalt(III) cation. When Co(NH3)6Cl3 titrations are performed on one mammalian and three bacterial DNAs, evidence is found for at least three distinct classes of bound Co(NH3)6(3+). A comparison of titration curves for all four DNAs demonstrates that an increase in GC content correlates with an increase in the fraction of specific Co(NH3)6(3+). binding sites. For M. lysodeikticus DNA (72% GC), a slowly exchanging class of bound 59Co(NH3)6(3+) is apparent. This class of sites is saturated at very low binding densities (between 0.02 and 0.03 cobalt cations per DNA phosphate). At higher binding densities (greater than 0.03), the signal due to slowly exchanging 59Co(NH3)6(3+) disappears into the noise, and a single 59Co(NH3)6(3+) signal is observed. Within the sensitivity limitations of these measurements, no evidence for slowly exchanging bound 59Co(NH3)6(3+) could be found for any of the other DNAs, for which a single, rapidly exchanging 59Co(NH3)6(3+) signal is observed at all binding densities. For this rapidly exchanging signal, for all four DNAs, the measured 59Co(NH3)6(3+) nmr parameters depend significantly on (a) binding density and (b) GC content of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase.

The Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two functionally and evolutionarily related enzymes with distinct substrate specificities. To understand the determinants of substrate specificities of the two proteins, the substrate and coenzyme in IPMDH were docked into their respective binding sites based on the published structure for apo IPMDH and its sequence and structural homology to ICDH. This modeling study suggests that (1) the substrate and coenzyme (NAD) binding modes of IPMDH are significantly different from those of ICDH, (2) the interactions between the substrates and coenzymes help explain the differences in substrate specificities of IPMDH and ICDH, and (3) binding of the substrate and coenzyme should induce a conformational change in the structure of IPMDH.     

Structure-function relationships in eukaryotic nuclei.

It may be that eukaryotic nuclei contain a collection of operationally independent units (genes), each controlled through its interactions with soluble protein factors which diffuse at random throughout the nucleoplasmic space. Alternatively, nuclei might be organized in such a sophisticated fashion that specific genes occupy distinct sites and that spatially ordered RNA synthesis, processing and transport delivers mature RNAs to predestined sites in the cytoplasm. Different fields of research support each of these extreme views. Molecular biologists inspecting the precise details of specific interactions, usually in vitro, inevitably favour the former, while cell biologists working with far more complicated systems generally assume that more elaborate arrangements exist. In considering the importance of nuclear architecture, I have attempted to relate a collection of experiments each of which intimates some close relationship between structural aspects of chromatin organization and the precise mechanisms underlying nuclear function. I will argue that higher-order structures are crucial for achieving the observed efficiency and coordination of many nuclear processes.     

Coenzyme interaction with horse liver alcohol dehydrogenase. Evidence for allosteric coenzyme binding sites from thermodynamic equilibrium studies.

The techniques of fluorescence enhancement, fluorescence quenching, fluorescence polarization, and equilibrium dialysis are utilized to study the binding properties of coenzyme to horse liver alcohol dehydrogenase. Polarization of fluorescence and equilibrium dialysis show that NADH binds to alcohol dehydrogenase with a stoichiometry of 6 mol per mol of enzyme, in contrast to the value of 2 determined from fluorescence enhancement measurements. NAD+ also binds with a stoichiometry of six as was determined by equilibrium dialysis. The two NADH sites which bind coenzyme more tightly and which are revealed by fluorescence enhancement measurements are designated the catalytic sites. Binding of coenzyme to the four ancillary sites does not alter the quantum yield of NADH but results in a 20% contribution to quenching of enzyme's tryptophan fluorescence. From the emission anisotropy of bound NADH of 24.0% for the additional sites and 28.1% for the catalytic sites and their relative fluorescence lifetimes at the same wavelengths of excitation and emmision, we conclude that the nicotinamide ring of NADH bound to the additional sites exhibits a freedom of motion independent of the macromolecule, while that bound to the catalytic sites is more rigidly held. Polarization of fluorescence yields negative intrinsic free energies of 9.2 and 7.5 Cal M-1 for NADH interaction with the catalytic and additional sites, respectively. Although these values are 1.3 to 2.0 Cal higher than those determined by fluorescence quenching and equilibrium dialysis, the mean Hill coefficient of 1.76 plus or minus 0.06, the titration span of 2.4 logarithmic units and coupling free energies (in magnitude and sign) are the same for all these techniques. The above difference in the intrinsic free energies are attributed largely to the different modes of interaction of excited and unexcited NADH molecules with alcohol dehydrogenase.     

Mannitol production in fungi during glucose catabolism.

The levels of phosphofructokinase (EC 2.7.1.11) and mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) have been determined in a number of Mucor and Penicillium species. Mannitol-1-phosphate dehydrogenase was found in only one species of mucor, Mucor rouxii, and this with a specific activity much lower than that found in Penicillium species. All of the fungi tested in the Ascomycetes class exhibited mannitol-1-phosphate dehydrogenase activity. Interference from both mannitol-1-phosphate dehydrogenase and NADH oxidase (EC 1.6.99.5) caused some difficulty initially in detecting phosphofructokinase in Penicillium species; the Penicillium phosphofructokinase is very unstable. Penicillium notatum accumulates mannitol intracellularly; detection of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase (EC 3.1.3.22) activity in cell-free extracts indicates that the mannitol is formed from glucose via fructose-6-phosphate and mannitol-1-phosphate; no direct reduction of fructose to mannitol could be detected. The mannitol-1-phosphate dehydrogenase was specific for mannitol-1-phosphate and fructose-6-phosphate; NADP+(H) could not replace NAD+(H). The phosphatase (EC3.1.3.22) exhibited a distinct preference for mannitol-1-phosphate as substrate; all other substrates tested exhibited less than 25% of the activity observed with mannitol-1-phosphate.     

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.