Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Measurement of the arylsulphatase of Patella vulgata with 4-methylumbelliferone sulphate

1. The preparation, purification, chemical and spectral properties of potassium 4-methylumbelliferone sulphate are described. 2. The use of 4-methylumbelliferone sulphate as a substrate for the arylsulphatase of Patella vulgata is presented with specific reference to the fluorimetric assay procedure used with this substrate. 3. 4-Methylumbelliferone sulphate is compared with the previously used synthetic sulphatase substrates nitrocatechol sulphate and p-nitrophenyl sulphate with respect to K_m, V_max. and sensitivity in the assay of arylsulphatase. 4. 4-Methylumbelliferone sulphate was strongly inhibited by phosphate. Sulphate, a less potent inhibitor, appeared to be of the competitive type with some anomalous characteristics.     

Adaptation of Drosophila enzymes to temperature IV. Natural selection at the alcohol-dehydrogenase locus

Studies were carried out on the temperature-dependent kinetic properties (K_m, Q₁₀, E_a, thermostability) of alcohol-dehydrogenase allozymes from D. melanogaster. It was shown that there is a parallelism between the biochemical properties of the enzymes and the behaviour of the genes in natural and cage populations. Furthermore, the relationship between the temperature-dependent kinetic properties of alcohol dehydrogenase and assay temperature was examined in three tropical and two temperate Drosophila species. K_m patterns were similar among species from the same habitat and different between habitats. No such parallelism was seen with respect to thermal inactivation. The Q₁₀ values in general reflected temperature-dependent changes in K_m. It is discussed that the mechanistic connection observed between the biochemical and population levels (intra-and interspecifically) strongly suggests that temperature acts as a selective factor on the structural Adh locus in the genus Drosophila.     

Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus sp.

Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O₂ to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn⁷⁵, Asn²³⁸, and Asn⁴⁵⁸ were elucidated. Initial rate kinetic analysis revealed that the k _cat, K _m, and k _cat/K _m of nLcc4 with substrate ABTS were 3,382 s ^-1, 65.0 ± 6.5 μM, and 52 s ^-1μM^-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s ^-1, 56.7 ± 3.2 μM, and 0.004 s ^-1μM^-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.     

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K_d, are 0.89 and 0.18 μM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.     

Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent-slope procedure: II. Application of the method to soluble and immobilized enzymes

A system is described for semiautomated evaluation of enzyme kinetic parameters, S_0.5, V, and Hill n. It consists of a Gilford spectrophotometer modified for continuous addition of substrate to a stirred enzyme assay mixture, and for recording of absorbance data coded on paper tape. The Hill parameters for a number of enzymes, obtained from these data by tangent-slope or curve-fitting procedures, are in good agreement with published or manually determined values. Experiments with rabbit muscle lactate dehydrogenase covalently linked to Sephadex G-50 or Sepharose 4B demonstrate the feasibility of this approach when applied to enzymes attached to solid supports. Instrumental errors due to spectrophotometer nonlinearity and drift, stirring, mixing, pumping, truncation in readout of absorbance, and bias in experimental parameters are minimal and do not significantly interfere with the method. The advantages and disadvantages of the approach are discussed, and improvements and extensions are suggested for the instrumental and data handling system which could greatly extend its utility.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Studies on the reaction mechanism of DT diaphorase. Action of dead-end inhibitors and effects of phospholipids.

The relationship between the inhibition of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC.1.6.99.2] by 4-hydroxycoumarin and the 1,3-indandione derivates was investigated. Evidence is presented that these two classes of anticoagulants, although both acting as competitive inhibitors with respect to NAD(P)H, bind to different sites of the enzyme in a synergistic fashion. These findings are interpreted as indicative of a cooperativity between different substrate-binding sites of the enzyme.Neutral phospholipids exert effects on partially purified rat-liver DT diaphorase similar to those earlier obtained with nonionic detergents. The effects concern several kinetic parameters of the enzyme, including V, K_m for electron donor and acceptor, and K_i for various inhibitors. The changes in the kinetic parameters vary in extent and direction according to the individual phospholipids.     

Reactivity of metal chelates of sulphur containing ligands towards Lewis bases. Part III. Reaction of bis(1,2-dimethyl-and 1-phenyl-1,2-ethanediylidene)bis(S-methylhydrazinecarbodithioate)NN′SS′(−2)dizinc(II) with pyridines

The reaction of the dimeric zinc(II) chelates of the type I (R₁ = R₂ = CH₃, R₁ = H, R₂ = Ph) with pyridine, 2-methylpyridine, 3-methylpyridine and 4-methylpyridine afforded the monomeric monobase adducts. The isolated adducts were characterized by their electronic and ¹H NMR spectra, and a five coordinate square pyramidal structure was tentatively assigned for these adducts.The adduct formation reaction was followed spectrophotometrically and the reaction kinetics were studied using a stopped flow technique. From the available kinetic data, as well as the measured activated parameters (ΔH^#, ΔS^#), a mechanism for the adduct formation reaction is proposed.     

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

Biphasic kinetic behavior of nitrate reductase from heterocystous, nitrogen-fixing cyanobacteria

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (K_m, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (K_m, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme.     

Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent-slope procedure: I. Analysis of the method using computed progress curves

A new method has been developed which provides reliable estimates of enzyme kinetic constants from single reaction progress curves recorded under conditions of continuously increasing substrate concentration. Equally spaced data points simulating such progress curves and containing known amounts of superimposed random noise were fit to the Hill equation by (i) direct nonlinear curve-fitting of raw data, and (ii) a tangent-slope technique in which the raw data are numerically differentiated, transformed into substrate versus velocity data, and then analyzed as linear plots. Both integral and differential procedures provided accurate and precise estimates of the Hill parameters (S_0.5, V, and n) from single reaction mixtures. However, the tangent-slope method was at least 10-fold faster to compute and was not dependent on accurate initial guesses of the Hill parameters or integration of the rate equation. With the tangent-slope method, the optimal number of data points used in calculating tangent slopes was found to be 9 or 11. The reliability of the Hill parameters determined with the tangent-slope method was relatively insensitive to the maximum substrate concentration over a range of $\text{S}{}_{\mathrm{<mtext>max</mtext>}}\text{S}{}_{0.5}$ of 1.5 to 10; the optimal value was 3. Through further analysis of simulated data, it was found that slow enzyme inactivation (<4% loss during the assay), or product competitive inhibition (maximum product concentration < 30% of the inhibitor dissociation constant) does not produce serious errors in the Hill parameters. Methods are presented to detect and distinguish enzyme inactivation and product competitive inhibition. It is suggested that continuous addition methodology combined with tangent-slope analysis provides the basis for a flexible system for kinetic characterization of enzymes which has wider applicability and other advantages over multicuvette or conventional progress curve methodology. A major advantage in contrast to the progress curve approach is that product accumulation and associated product effects are lowest at lower substrate concentrations.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata

Environmental factors that shape dynamics of benthic toxic blooms are largely unknown. In particular, for the toxic dinoflagellate Ostreopsis cf. ovata, the importance of the availability of nutrients and the contribution of the inorganic and organic pools to growth need to be quantified in marine coastal environments. The present study aimed at characterizing N-uptake of dissolved inorganic and organic sources by O. cf. ovata cells, using the ¹⁵N-labelling technique. Experiments were conducted taking into account potential interactions between nutrient uptake systems as well as variations with the diel cycle. Uptake abilities of O. cf. ovata were parameterized for ammonium (NH₄⁺), nitrate (NO₃⁻) and N-urea, from the estimation of kinetic and inhibition parameters. In the range of 0 to 10 μmol N L⁻¹, kinetic curves showed a clear preference pattern following the ranking NH₄⁺ > NO₃⁻ > N-urea, where the preferential uptake of NH₄⁺ relative to NO₃⁻ was accentuated by an inhibitory effect of NH₄⁺ concentration on NO₃⁻ uptake capabilities. Conversely, under high nutrient concentrations, the preference for NH₄⁺ relative to NO₃⁻ was largely reduced, probably because of the existence of a low-affinity high capacity inducible NO₃⁻ uptake system. Ability to take up nutrients in darkness could not be defined as a competitive advantage for O. cf. ovata. Species competitiveness can also be defined from nutrient uptake kinetic parameters. A strong affinity for NH₄⁺ was observed for O. cf. ovata cells that may partly explain the success of this toxic species during the summer season in the Bay of Villefranche-sur-mer (France).     

A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.     

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet–SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k_cat, K_M, and catalytic efficiency (k_cat/K_M) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.     

Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, K_m(ATP) 0.60mm, K_m(glucose) 0.042mm; peak II, K_m(ATP) 0.66mm, K_m(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, K_m(ATP) 0.56mm, K_m(glucose) 0.048mm; peak II, K_m(ATP) 0.68mm, K_m(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.     

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.     

Kinetic properties of phosphoenolpyruvate carboxylase from c(3), c(4), and c(3)-c(4) intermediate species of flaveria (asteraceae)

Flaveria cronquistii (C₃), F. chloraefolia (C₃-C₄), F. floridana (C₃-C₄), F. pubescens (C₃-C₄), F. anomala (C₃-C₄), F. linearis (C₃-C₄), F. brownii (C₄), F. palmeri (C₄), F. trinervia (C₄) and F. australasica (C₄), comprising 10 out of the 21 known species of the genus Flaveria (Asteraceae), were included in a comparative study of the kinetic and regulatory properties of green leaf phosphoenolpyruvate (PEP) carboxylase. At least three kinetically distinct enzyme-forms were identified on the basis of their affinities for PEP and the degree of allosterism with respect to this substrate. The kinetic properties of PEP carboxylase of most of the species seemingly were modified in vivo depending on the growth conditions of the plants. K_m(PEP_free)-values of the enzyme from the five C₃-C₄ intermediate species ranged from 6 micromolar (F. chloraefolia, low light-grown) to 38 micromolar (F. pubescens, high light-grown). In contrast, the K_m for PEP of PEP carboxylase from the C₃ species F. cronquistii (13 micromolar) apparently was not influenced by growth conditions. The response of the enzyme from the C₃ and C₃-C₄ species was hyperbolic in all cases. A second isoform with a lower affinity for PEP (88-100 micromolar), but also hyperbolic kinetics was found in the C₄ species F. brownii, whereas in the three other C₄ species examined a PEP carboxylase with a still lower affinity for PEP (187-221 micromolar) and sigmoidal kinetics was present. These isozyme-related kinetic data were supported by analyses of the elution behavior of the enzyme during anion-exchange chromatography on DEAE-Trisacryl M. The results are discussed with respect to the evolution of C₄ photosynthesis in the Flaveria genus.     

Structure-function studies on gastrointestinal hormones: I. Synthesis of secretin analogs and their biological and immunological properties

Syntheses by conventional procedures of the three analogs corresponding to the porcine secretin sequence crossed at position 6 by the N-terminal hexapeptide sequences of VIP, GIP, and glucagon are described, viz., Ala⁴,Val⁵-, Tyr¹,Ala²,Glu³-, and Gln³-secretin (VIP-SN, GIP-SN, and GLU-SN). The analog Phe¹,Phe²,Trp³,Lys⁴-secretin (SOMA-SN), designed on the basis of the surprising homology of the sequence portions 10–13 of somatostatin and 5–8 of secretin, was also prepared. Finally, the synthesis of N^α-3-(4-hydroxyphenyl)propionyl-β-alanyl-secretin (DATA-SN), a tracer suitable for secretin radioimmunoassay and as an N-terminus modified secretin analog, is reported. The analogs are compared, in terms of their biological and immunological properties in different assay systems, with pure synthetic secretin.