Occupation Of Active Sites Research Articles

Activation and Active Site Occupation Alter Conformation in the Region of the First Epidermal Growth Factor-like Domain of Human Factor VII

The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.

Probing the Active Site of the Hepatitis C Virus Serine Protease by Fluorescence Resonance Energy Transfer

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

Crystal Structure of Active Site-Inhibited Human Coagulation Factor VIIa (des-Gla)

Factor VIIa (FVIIa) is a crucial haemostatic protease consisting of four distinct domains termed the Gla, epidermal growth factor-1 (EGF-1), EGF-2, and protease domains (from N- to C-terminus). The crystal structure of human FVIIa inhibited at the active site with 1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone and lacking the Gla domain has been solved to a resolution of 2.28 Å. The EGF-2 and protease domains were well resolved, whereas no electron density for the EGF-1 domain was observed, suggesting a flexible arrangement or disorder within the crystal. Superposition of the protease domain of the present structure with that previously resolved in the tissue factor (TF)/FVIIai complex revealed that although overall the domain structures are similar, the EGF-2 domain is rotated by 7.5° relative to the protease domain on binding TF. A single cleavage in the protease domain was found, between Arg315 and Lys316 (chymotrypsin numbering 170C–170D) in a FVII-specific insertion loop: this cleavage appeared to be essential for crystallisation. Insertion of the heavy chain N-terminal Ile153 is essentially identical in the two structures, as is the geometry of the active site residues and the inhibitor C-terminal arginine residue. Some differences are seen in the cleaved loop, but changes in TF-contact residues are generally minor. This structure supports the hypothesis that TF binding enables spatial domain arrangements in the flexible FVIIa molecule necessary for procoagulant function and furthermore that active site occupancy induces FVIIa active conformation via N-terminal insertion.

Role of the cofactor calcium in the activation of outer membrane phospholipase A.

The enzymatic activity of the outer membrane phospholipase A (OMPLA), an integral membrane protein of Escherichia coli, is regulated by dimerization for which the cofactor Ca2+ is required. In this study, the interaction of Ca2+ with OMPLA was characterized, with an emphasis on the role of the cofactor in the activation process and dimerization. Kinetic experiments were done in which the enzyme was solubilized in mixed micelles of substrate and different detergents. It appeared that the affinity of OMPLA for Ca2+ was high (12 microM) if alkylphosphocholines were used as detergent, moderate (62 microM) if sulfobetaines were used, and very low (24 mM) if alkylpolyoxyethylene glycols were used. These results show that there is a strong modulation of the calcium binding properties of OMPLA by the lipid environment. In the presence of hexadecylphosphocholine micelles, the affinity of OMPLA for Ca2+ was determined by three direct binding techniques. Using gel filtration, it appeared that OMPLA has one high-affinity site (Kd approximately 36 microM) and a second site with moderate affinity (Kd approximately 358 microM). Sulfonylated-OMPLA, in which the active site serine had been covalently modified with hexadecanesulfonylfluoride, was used as a mimic for the acyl-enzyme intermediate. In gel filtration experiments, this sulfonylated-OMPLA displayed binding of two Ca2+ per enzyme monomer both with similar high affinity (Kd approximately 48 microM), indicative of a strong synergistic effect of active site occupation and the affinity of the second Ca2+ binding site. Isothermal titration calorimetric measurements confirmed only the presence of a high-affinity Ca2+ binding site, whereas in fluorescence experiments only the binding of the second Ca2+ could be observed. Chemical cross-linking was applied to investigate which of the two Ca2+ sites is involved in dimerization. OMPLA was monomeric in the absence of Ca2+, whereas already at low Ca2+ concentrations the enzyme was converted to its dimeric form. Therefore, we suggest that the first Ca2+ plays a role in the stabilization of the dimeric state of the enzyme. The role of the second Ca2+ and the observed synergy between active site occupancy and Ca2+ affinity are discussed.

Influence of cofactor binding and active site occupancy on the conformation of the macromolecular substrate exosite of factor VIIa

The catalytic activity of the trypsin-like serine protease coagulation factor VIIa is allosterically regulated. In this work, we employed monoclonal antibodies as probes to analyze conformational changes in the VII protease domain that are induced by zymogen activation, cofactor tissue factor (TF) binding, and active site occupancy. The epitopes of three monoclonal antibodies were mapped using a panel of 57 individual alanine replacement mutants in the protease domain. Two of the antibodies had typical “hot spot” epitopes in a basic cluster above the active site cleft and antibody binding to these epitopes was not affected by zymogen activation, TF binding, or active site occupancy. In contrast, the binding kinetics of VII/VIIa to a monoclonal antibody that mapped to an extended epitope overlapping with the macromolecular substrate exosite was affected by each of the conformational transitions of the VIIa protease domain. The changes in antibody affinity are consistent with a transition from zymogen VII to the TF·VIIa complex, with free enzyme VIIa as an intermediate that retains some zymogen-like features responsible for its low catalytic activity. In contrast, active site occupancy resulted in effects that were qualitatively different from the effects of zymogen activation on the antibody epitope. This provides novel insight into the conformational interdependence between the active site, the region for macromolecular substrate recognition, and the cofactor binding exosite of this allosterically regulated serine protease.

Active Site Modification of Factor VIIa Affects Interactions of the Protease Domain with Tissue Factor

In the initiation of coagulation, tissue factor (TF) allosterically activates the serine protease factor VIIa (VIIa) through specific interactions with protease domain residues. These interactions, and consequently affinity for TF, may be influenced by conformational changes in the protease domain that result from zymogen-enzyme transition or occupancy of the active site by tight binding inhibitors. In functional competition and direct binding analysis, we determined affinities for zymogen and enzyme species of wild-type VII and of mutants at protease domain residues that contact TF. We demonstrate that TF binding is not influenced by zymogen activation, indicating that the protease domain of zymogen and enzyme dock similarly with TF. In contrast, active site occupancy enhanced the affinity for TF by predominantly decreasing the dissociation rate of the TF.VIIa complex. Of the three interface residues studied, only Met306 played a major role in the inhibitor-induced increase in affinity. Met306 is also important for transmitting the allosteric changes from TF to the active site, resulting in enhanced catalysis. This study thus provides evidence for a bidirectional conformational interdependence of the interface residue Met306 and the active site of VIIa.

N2O in NO Reduction by CO Over Pt/NaX Zeolite

Pt/NaX zeolite (3 Pt atoms per unit cell) was prepared by vacuum decomposition of [Pt(NH3)4]2+. NO + 13CO and N2O + 13CO reactions were studied under static conditions at 180, 205 and 230 °C and followed by temperature programmed desorption (TPD) of surface species adsorbed in zeolites during the reaction. The effect of different NO/CO ratios and of the added oxygen was examined for the former reaction. The experimental results agree with the assumption that N2O (and 13CO2) is (are) the primary product(s) released into the gaseous phase below 230 °C; above 205 °C, the complete reduction to N2 occurs. N2O could be a very rapidly decomposing surface intermediate for this complete reduction, and can act together with the recombination of N atoms. Nitrous oxide released into the gas phase cannot serve as the reaction intermediate, because its reduction by CO proceeds much more slowly than that of NO. The formation of nitrous oxide in the NO + CO reaction is, in addition to low temperatures, enhanced by the increased NO/CO ratio and by the presence of oxygen. The latter effect can be due to the occupation of Pt active sites by adsorbed oxygen and/or by the oxidation of NO to higher oxides which decompose, yielding N2O (together with NO and oxygen).

Selective Hydrogenation of Phenylacetylene on Pumice-Supported Palladium Catalysts

The liquid phase, selective hydrogenation of phenylacetylene on pumice-supported palladium catalysts has been studied for a large range of metallic dispersions (14% ≤ D x ≤ to 62%). The kinetics were analyzed by a five-parameter mathematical model. The mechanism was determined by the contribution of three basic routes involving only surface species in the rate-determining steps. The hydrogenation of phenylacetylene to styrene is "structure insensitive." The disappearance rate constant of styrene produced from phenylacetylene is slightly lower than that of phenylacetylene and does not change in the case of the direct hydrogenation of styrene on the same Pd/pumice catalyst. However, Q 3 (the ratio of adsorption constants K Eb/ K St, where Eb is ethylbenzene and St is styrene), which is typical of a zero order reaction ( Q 3 → 0) in the case of the direct hydrogenation, is practically constant ( Q 3 ≅ 2) in the case of hydrogenation of styrene produced from phenylacetylene. This is explained by the formation, in the latter case, of polymeric species or other species which are difficult to hydrogenate and by the consequent occupation of active sites so that the adsorption of styrene is inhibited. These species are also thought to be responsible for a flattening effect in the catalytic activity. Activity and selectivity data are critically analyzed and compared with those reported for other supported palladium catalysts. Since Pd/pumice catalysts also show high activity and selectivity at high metal dispersions, they could be of interest for industrial applications.

Effects of linear alkylbenzene sulfonate (LAS) on the adsorption behaviour of phenanthrene on soils

Experimental measurements of adsorption of phenanthrene were made at 25°C with and without the presence of linear alkylbenzene sulfonate (LAS) on two soils from Shenyang, P. R. China and LUFA, Speyer, Germany. Effects of LAS on the adsorption of phenanthrene were studied in LAS concentration range around its critical micelle concentration (cmc) with emphasis on low LAS levels which are relevant to the environment. Two methods were used to simulate different environmental conditions (contacting sequence of soils with test chemicals): “soil+phenanthrene+LAS” and “soil+LAS+phenanthrene”. The contacting sequences were found to have significant influence on phenanthrene adsorption at low LAS levels <50 μm ml −1. The combination of “soil+phenanthrene+LAS” resulted in an increase in phenanthrene adsorption probably due to partition of phenanthrene to LAS hemimicelles/admicelles formed on the soil surface, while the combination of “soil+LAS+ phenanthrene” decreased the adsorption because of occupation of active hydrophobic adsorption sites by LAS. In the presence of higher concentrations (>50 μm ml −1) of LAS, results obtained with both methods indicated that adsorption of phenanthrene decreased. The values of K d decreased from 250.7 in the absence of LAS to 112.3 and 31.9 in the presence of 500 μg ml −1 and 1000 μg ml −1 LAS, respectively. Solubilization of phenanthrene by LAS may be the dominant effects that cause the decrease of phenanthrene adsorption at high LAS concentrations.

Cloning, expression and mutational analysis of the urocanase gene (hutU) fromPseudomonas putida

The histidine-utilizing hutU gene was isolated from a lambda-EMBL3 phage of a genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7-7. Escherichia coli BL21 cells were transformed with the recombinant plasmid and produced a catalytically active protein, amounting to approximately 30% of the total protein in the crude cell-free extract. The addition of NAD+ to the growth medium ensured the full occupation of active sites by the cofactor. This requires a mechanism for the transport of NAD+ into E. coli cells. Using the overproducing mutant a new, fast and efficient isolation procedure is described which yields electrophoretically homogeneous urocanase within two days. The yield of pure enzyme, based on the culture volume, has been improved 50-80-fold compared with the traditional method. To investigate the possible role of cysteine residues in the catalysis or in the tight binding of the cofactor NAD+, six different mutants were prepared. In each mutant protein, one conserved cysteine was exchanged for alanine. The resulting clones were tested for the expression of urocanase with catalytic activity; the Km and Vmax values were determined. Only Cys410 was essential for catalysis. There was no detectable reconstitution or increase of activity after the addition of NAD+, either in the essential Cys/Ala mutant or the other mutant proteins. Electrospray-mass spectroscopy of the wild-type enzyme revealed that the coenzyme is not covalently bound to the protein and computational analysis showed no typical sequence for a mononucleotide-binding domain like the Rossman fold. To obtain urocanase apoenzyme, P. putida nicII was transformed with pGP1-2 and pTET7-U and grown in nicotinate-depleted medium. Like the mutant proteins, no activation of the apoform occurred after the addition of NAD+. These observations led us to postulate a new model for the non-covalent but tight binding of NAD+ to the enzyme by 'trapping' the cofactor while folding the nascent protein.

Dehydrogeno-aromatization of cyclohexanes with suspended noble-metal catalysts

Under boiling and refluxing conditions, suspended noble-metal catalysts produced selective dehydrogeno-aromatization of cyclohexanes, yielding aromatic compounds and molecular hydrogen with Pt being more active than Ru. The retardation effects caused by aromatic compounds being present in the solution were ascribed not only to active-site occupation but also to hydrogen transfer, which consumed the surface hydrogen species as the precursor of molecular hydrogen.

Kinetic‐Thermodynamic and Adsorption Isotherms Analyses for the Inhibition of the Acid Corrosion of Steel by Cyclic and Open‐Chain Amines

The corrosion inhibition characteristics of the quadridentate macrocycle 1, 4, 8, 11 tetra‐azacyclotetradecane (cyclam) on steel in sulfuric acid media are investigated at 25, 35, 45, and 55°C, and the thermodynamic energy parameters calculated. In addition the inhibition characteristics of the open‐chain polydentate amines, ethylenediamine (en), diethylenetriamine (dien), triethylenetetraamine (trien), and tetraethylenepentamine (tetren) were investigated at 25°C. Activation‐free energies, enthalpies, and entropies for the inhibition process of both cyclam and tetren were determined from the rate constants data measured at four temperatures viz., 25, 35, 45, and 55°C at one or two fixed inhibitor concentrations. All data were compared and fitted to a previously published kinetic‐thermoydnamic model in terms of an active site occupancy parameter and a binding constant of the inhibitor to the active site. The results obtained are compared with fits obtained from the application of Fumkin and Flory‐Huggins adsorption isotherms. The first isotherm attempts to treat the deviations from the ideal Langmuir isotherm by the use of a lateral interaction parameter while the second uses a relative‐size parameter which is a measure of the relative size of the inhibitor molecule to the surface‐adsorbed water molecule. Good agreement was obtained between the kinetic‐thermodynamic and Flory‐Huggins models. The Frumkin isotherm model, however, agreed only in cases where the active site occupancy, or the relative‐size parameters were equal to or close to unity. A discussion of the strengths and weaknesses of each treatment is given. In addition, the results obtained are correlated to the chemical structure of the inhibitors, the number of donor atoms, and the macrocyclic effect.

Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli.

Anaerobically grown Escherichia coli contain an enzymatically active iron superoxide dismutase (Fe2-FeSOD) and an inactive iron-substituted manganese superoxide dismutase (Fe2-MnSOD). The anaerobic electron sink, nitrate plus paraquat, enhanced biosynthesis of the MnSOD polypeptide, with accumulation of inactive Fe2-MnSOD. The oxidant, diamide, in contrast, allowed anaerobic production of the active forms of MnSOD, i.e. Mn2-MnSOD and Mn/Fe-MnSOD. Nutritional supplementation with Mn(II) favored occupancy of the MnSOD active site with manganese and allowed anaerobic accumulation of Mn2-MnSOD in the absence of diamide. Enrichment of the anaerobic growth medium with Fe(II) both suppressed biosynthesis of the MnSOD polypeptide and inhibited formation of the active manganese-containing forms. A tac-sodA operon fusion was used to examine the effects of chelating agents and metals on maturation of nascent MnSOD, independent from the transcriptional effects these agents impose. Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) elicited anaerobic biosynthesis of MnSOD, which accumulated as the inactive Fe2-MnSOD. Diamide, with IPTG, allowed formation of active Mn/Fe-MnSOD while 1,10-phenanthroline with IPTG resulted in accumulation of Mn2-MnSOD. These results suggest that iron participates in the redox-sensitive control of the formation of active MnSOD at two levels, i.e. that of transcription as well as that of maturation. During maturation of the nascent MnSOD polypeptide, iron and manganese compete for the metal-binding site; anaerobic conditions favor iron-binding, whereas oxidants, such as dioxygen or diamide, favor binding of manganese.

Reversed-phase high-performance liquid chromatographic—radioreceptor assay of human cerebrospinal fluid neuropeptides: Maximizing recovery of picomoles of peptides and minimizing memory

Reversed-phase high-performance liquid chromatography (RP-HPLC) is used to analyze neuropeptides in human cerebrospinal fluid (CSF) obtained from patients suffering from lower-back pain. Because CSF contains only femtomole to picomole amounts of those peptides, it is important to minimize any sample memory effect, while maximizing peptide recovery and avoiding any potential artifactual peak formation during chromatography. This study describes the phenomenon of active site occupancy by peptides on the RP-HPLC column, which is crucial when studying CSF where femtomole to picomole amounts of neuropeptides could be lost. Knowledge of those basic Chromatographic factors is important whenever biologic extracts of peptides are applied to and eluted from an RP-HPLC column and then detected off-line with a radioreceptor assay, which is sensitive at the picomole level.

Proteolytic inactivation of human leukocyte elastase.

Human leukocyte elastase can be proteolytically inactivated by bovine pancreatic trypsin. Neither porcine pancreatic elastase nor bovine pancreatic chymotrypsin causes inactivation of leukocyte elastase, nor are trypsin, pancreatic elastase, or chymotrypsin themselves susceptible to proteolysis. The trypsin-catalyzed inactivation of leukocyte elastase can be slowed by inhibition of trypsin with benzamidine or by occupation of elastase's active site with elastatinal.

Coenzyme active site occupancy as an indicator of independence of the subunits of mitochondrial aspartate aminotransferase.

The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which contain overlapping subunit regions ( Eichele , G., Ford, G.C., Glor , M., Jansonius , J.N., Mavrides , C., and Christen , P. (1979) J. Mol. Biol. 133, 161-180), suggesting the possibility of subunit interactions. The structurally similar cytosolic isozyme exhibits noncooperative binding of pyridoxal 5'-phosphate ( Boettcher , M., and Martinez -Carrion, M. (1975) Biochemistry 14, 4528-4531; Relimpio , A., Iriarte , A., Chlebowski , J.F., and Martinez -Carrion, M. (1981) J. Biol. Chem. 256, 4478-4488) in which the apoenzyme/holoenzyme hybrid dimer shows a distinctive thermal stability. Using a nonequilibrium isoelectric focusing technique, it can be shown that mitochondrial aspartate aminotransferase also binds cofactor in a noncooperative random fashion. However, differential scanning calorimetry (DSC) thermograms show different characteristics from the cytosolic form. These differences are interpreted in terms of unique subunit interactions in this isozyme. Heating to the various DSC transition temperatures shows that the anomalous DSC thermograms in partially coenzyme-saturated apoenzyme preparations are due to a selective dissociation of apoenzyme subunits into monomers which are irreversibly denatured. The remaining holoenzyme monomers reassociate and form stable holoenzyme dimers. The net result is retention of the initial concentration of holoenzyme subunits present in any given mixture. Random occupancy of active sites and similar electrophoretic and DSC patterns upon heating of partially saturated apoenzyme preparations is observed whether the coenzyme, pyridoxal phosphate or pyridoxamine phosphate alone, or borohydride-reduced Schiff's bases of coenzyme-substrate analogue derivatives are used as active site directed ligands. The latter resemble covalent enzyme-substrate intermediates.

Evidence for inner-sphere coordinated alkoxide ion intermediates in the catalytic mechanism of Co(II) substituted liver alcohol dehydrogenase

Substitution of certain divalent transition metal ions, i.e. Co(II), Ni(II), Cu(II) and Fe(II), for the active site ion of horse liver alcohol dehydrogenase yields catalytically active enzyme derivatives [1–4]. Since catalysis occurs via inner-sphere coordination of substrate [3,5,6], the ligand-to-metal charge-transfer (LMCT) and d-d electronic transitions of these metal ions are useful spectroscopic probes of the chemical events occurring during catalysis [1, 2, 7]. As will be shown in this report, when reaction is monitored via rapid-scanning, stopped-flow (RSSF) UV-visible spectroscopy, the resulting time-resolved spectra (3000 nm to 700 nm) provide detailed temporal information about transient intermediates formed during catalysis. We report here evidence from RSSF spectroscopy for the existence of ternary enzymeNAD+alkozide ion intermediates both in the Co(II)E-catalyzed reduction of aldehydes and in the Co(II)E-catalyzed oxidation of alcohols.

Phenylalanyl-tRNA synthetase of baker's yeast. Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid.

Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic [32P]pyrophosphate (ATP--[32P]PPi) exchange activity via inhibition of adenylate synthesis. Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating. In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate. At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated. Occupation by these reactants is assumed to follow homotropic negative cooperativity. The type of effects depends on modification of tRNAPhe. Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator. Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride. Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA. In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.

Synthesis and quality control of aliphatic and aromatic 18F-labelled compounds for probing metabolism in-vivo

Biomolecules tagged with fluorine-18, a positron emitter with a half-life of 110 min, are gaining importance in diagnostic nuclear medicine for measuring regional functions in-vivo by means of positron emission tomography. Procedures for introducing 18 F into organic compounds, however, are limited due to the short half-life. In addition, the toxicity of many fluorine compounds requires practically carrier-free products. Hence, fast syntheses have to be carried out using fluorinating agents in the micro- or nanogram scale. On the other hand, the tracer provides unique possibilities for studying metabolic functions of toxic or centrally active fluorine compounds in-vivo. We have synthesized several aliphatic and aromatic fluorine-18 labelled compounds by nucleophilic 18 F-for-halogen exchange: For the study of regional metabolism in heart and liver of mice 16- 18 F-hexadecanoic acid, 17- 18 F-heptadecanoic acid, 2- 18 F-, and (9,10)- 18 F-stearic acid were prepared in a mixture of molten acetamide and the corresponding bromofatty acid ester followed by hydrolysis and purification by high pressure liquid chromatography. Variation of temperature, reaction time, and KF- carrier finally led to an optimum radiochemical yield of about 30% [1]. The biochemical effects of the fluorine label, as expected on the basis of β-oxidation, is clearly reflected in the pharmacokinetics and the chemical fate of the fluorine label observed in mice: The odd-numbered compound, 17- 18 F- heptadecanoic acid, is catabolized to β- 18 F-propionic acid while the even-numbered 16- 18 F-hexadecanoic acid ends up with 18 F-fluoroacetic acid entering the citric cycle. Further degradation, i.e. dehalogenation, only occurs in the case of 17- 18 F-heptadecanoic acid yielding free 18 F- fluoride which can be detected in high yield among its metabolites [2]. With respect to diagnostic methods for the localisation of thrombi, the protein urokinase labelled with 18 F- fluoroacetic acid could be a useful compound, since it is expected that it will be concentrated in the thrombus thus giving the possibility of localisation by positron- emission tomography. 18 F-fluoroacetic acid has therefore been prepared carrier-free in order to prevent the occupation of active sites in urokinase. For the study of regional metabolism in brain, 2- 18 F- nicotinic acid diethylamide is potentially useful. The nonhalogenated compound is known to be a centrally acting pharmaceutical. Preparation of the 18 F-labelled compound was carried out by a similar procedure as in the case of the fatty acids starting from the corresponding chloro- compound. By this method optimum radiochemical yields up to 46% could be obtained. Preliminary results in experiments with mice show a fast accumulation of 18 F-activity in the brain within the first seconds after injection, followed by a slower decrease [3].

Synthesis and use of bifunctional chloromethylalkanedione derivatives of variable chain length for cross-linking thiol groups in oligomeric proteins. Specific cross-linking in glyceraldehyde 3-phosphate dehydrogenase

Bischloromethylpentanedione, bischloromethylhexanedione, bischloromethyloctanedione and bischloromethyldecanedione were synthesized from their corresponding dicarboxylic acids via the bis-acyl chloride and the bisdiazomethylketone derivatives. These compounds proved to be highly specific cross-linking reagents for rabbit skeletal-muscle glyceraldehyde 3-phosphate dehydrogenase. Incubation of the enzyme with cross-linking reagents resulted in both a time- and concentration-dependent formation of covalently linked oligomeric structures. The major cross-linked product detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was the dimer (mol. wt. 72000). Sepharose 6B chromatography of the cross-linked enzyme showed that it still existed as the tetramer. Cross-linking was dependent on the native structure of the enzyme, since it was abolished on denaturation of the enzyme. The actual covalently linked product depends on the conditions of modification and the chain length of the reagent. The maximum yield of dimer (70-80%) was obtained with bischloromethylhexanedione, and the yield decreased with either shorter- or longer-chain compounds. The calculated distance between the two reactive points in bischloromethylhexanedione is 1.21-1.45nm. Bischloromethylhexanedione modified at least two thiol groups per monomer. Modification of the active-site thiol, cysteine-149, was not essential for cross-linking, since glyceraldehyde 3-phosphate dehydrogenase carboxymethylated on cysteine-149 still reacted to form the dimer. The rate of chemical cross-linking was markedly decreased by increasing the NAD(+) occupancy of the enzyme active sites. These experiments are discussed in terms of the asymmetry of the enzyme structure in solution.