Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity

A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified ¹²⁵I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor.     

Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants: protein purification from corn leaves and immunological detection

We have purified the protein for the enzyme sucrose phosphate synthase (SPS) from corn (Zea mays) leaves. Partially purified SPS protein was used to generate specific monoclonal antibodies. The following immunoaffinity chromatography allowed the isolation of pure SPS protein. The apparent molecular mass of the SPS polypeptide is 138 kilodaltons. By immunoblot, an SPS antigen was found to accumulate in mature leaves. SPS protein levels remain constant during the day/night cycle. The observed diurnal fluctuation of extractable enzyme activity, therefore, must be caused by modification of the specific activity of SPS in vivo.     

Involvement of protein dynamics in enzyme stability. The case of glucose oxidase

Dynamics of glucose oxidase immobilized and in solution were compared through their tryptophan fluorescence spectra, decay times and quenching by acrylamide. Energy barrier for thermal inactivation and melting temperature of both soluble and immobilized enzyme were also measured. Data show that the fluctuation amplitude is at the origin of protein instability.     

Polyamines and ornithine decarboxylase activity during growth and differentiation in Blastocladiella emersonii.

The activity of ornithine decarboxylase (EC 4.1.1.17, L-ornithine carboxy-lyase) was determined during the life cycle of Blastocladiella emersonii. The specific activity of the enzyme was found to be low in the zoospores, to rise 20-fold during germination and early growth, to fall during growth and to rise again during sporulation. This rise in enzyme activity was shown to be dependent on protein synthesis. Putrescine levels, on a per mg of protein basis, paralleled the fluctuation found in ornithine decarboxylase activity. Putrescine and spermidine were the only polyamines found in extracts of B. emersonii.     

Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of dopa-responsive dystonia

To explore the molecular etiology of two disorders caused by a defect in GTP cyclohydrolase I--hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency--we purified and analyzed recombinant human wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant-negative effect of chimeric protein composed of wild-type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD.     

Hydrogen-deuterium exchange studies on guanidinated pig heart lactate dehydrogenase

Pig heart lactate dehydrogenase becomes more thermostable on increasing the degree of guanidination (conversion of lysine to homoarginine) (Minotani, N., Sekiguchi, T., Bautista, J.G. and Nosoh, Y. (1979) Biochim. Biophys. Acta 581, 334-341). The conformational change of the protein on guanidination was then examined by hydrogen-deuterium (H-2H) exchange reactions. It ws found that (i) the fluctuation degrees of peptides and tyrosine and tryptophan residues in the protein decrease in that order, (ii) two H-2H exchangeable tryptophan residues per subunit are freely accessible to solvent and the fluctuation degrees of the residues does not change on guanidination, (iii) the H-2H exchange detectable tyrosine residues are not freely accessible to solvent and become less fluctuating when 15 lysine residues per subunit are guanidinated, and (iv) the peptides become much less fluctuating on increasing the degree of guanidination. The specific activity of the enzyme decreased on guanidination. The increased thermostability of the protein on guanidination may be related to the decrease in flexibility of the molecular structure by sacrificing the enzyme activity.     

NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar

Conformational fluctuation plays a key role in protein function, but we know little about the associated structural changes. Here we present a general method for elucidating, at the atomic level, a large-scale shape change of a protein molecule in solution undergoing conformational fluctuation. The method utilizes the intimate relationship between conformation and partial molar volume and determines three-dimensional structures of a protein at different pressures using variable pressure NMR technique, whereby NOE distance and torsion angle constraints are used to create average coordinates. Ubiquitin (pH 4.6 at 20 degrees C) was chosen as the first target, for which structures were determined at 30 bar and at 3 kbar, giving "NMR snapshots" of a fluctuating protein structure at atomic resolution. The result reveals that the helix swings in and out by >3 angstroms with a simultaneous reorientation of the C-terminal segment, providing an "open" conformer suitable for enzyme recognition. Spin relaxation analysis indicates that this fluctuation occurs in the ten microsecond time range with activation volumes -4.2(+/-3.2) and 18.5(+/-3.0) ml/mol for the "closed-to-open" and the "open-to-closed" transitions, respectively.     

Temperature-induced change in the Ca2+-dependent ATPase activity and in the state of the ATPase protein of sarcoplasmic reticulum membrane.

The temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18 degrees C for all the preparations. Hydrogen--deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18 degrees C for all the preparations. It is concluded that the break at around 18 degrees C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.     

Expression of P-Enolpyruvate Carboxylase and other Aspects of CAM during the Development of Peperomia camptotricha Leaves

During the development of Peperomia camptotricha leaves, metabolism changes from C₃-photosynthesis to Crassulacean acid metabolism (CAM). The youngest leaves showed no diurnal fluctuation of organic acids or P-enolpyruvate carboxylase (PEPc) activity. There was little evidence for PEPc protein using PEPc antibodies prepared from the CAM form of PEPc, nor was there evidence for PEPc mRNA when tested using a cDNA probe made from CAM P. scandens. As leaves matured, there was a parallel increase in titratable acidity, PEPc activity, PEPc protein, and PEPc mRNA. In leaf whorls 1 through 6, there was a significant linear correlation between the diurnal fluctuation of organic acids and PEPc activity indicating a functional relationship. The specific activity of PEPc increased as leaves matured and the K_m (PEP) decreased indicating that the enzyme was becoming more active. The ratio of PEPc protein to PEPc mRNA decreased as leaves matured. During the expression of CAM, the spongy mesophyll where most of the CAM activity occurs increased in thickness and per cent air space, whereas the palisade mesophyll where most of the C₃ activity occurs did not increase in size dramatically. The diurnal fluctuation of organic acids and the expression of PEPc activity, protein, and mRNA increased as the thickness of the spongy mesophyll increased. During the expression of CAM in Peperomia camptotricha, there appears to be coordinated expression of PEPc mRNA, protein, and activity, the commencement of diurnal organic acid fluctuation, and the development of the CAM-like spongy mesophyll. Thus the evidence suggests that CAM in this species is expressed during normal development and not in response to environmental signals.     

Proteins with similar architecture exhibit similar large-scale dynamic behavior

We have investigated the similarities and differences in the computed dynamic fluctuations exhibited by six members of a protein fold family with a coarse-grained Gaussian network model. Specifically, we consider the cofactor binding fragment of CysB; the lysine/arginine/ornithine-binding protein (LAO); the enzyme porphobilinogen deaminase (PBGD); the ribose-binding protein (RBP); the N-terminal lobe of ovotransferrin in apo-form (apo-OVOT); and the leucine/isoleucine/valine-binding protein (LIVBP). All have domains that resemble a Rossmann fold, but there are also some significant differences. Results indicate that similar global dynamic behavior is preserved for the members of a fold family, and that differences usually occur in regions only where specific function is localized. The present work is a computational demonstration that the scaffold of a protein fold may be utilized for diverse purposes. LAO requires a bound ligand before it conforms to the large-scale fluctuation behavior of the three other members of the family, CysB, PBGD, and RBP, all of which contain a substrate (cofactor) at the active site cleft. The dynamics of the ligand-free enzymes LIVBP and apo-OVOT, on the other hand, concur with that of unliganded LAO. The present results suggest that it is possible to construct structure alignments based on dynamic fluctuation behavior.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : I. CO2 Fixation and Ribulose-1,5-Diphosphate Carboxylase Synthesis

A mutant strain of the green alga Chlamydomonas reinhardi, ac-20, is described in which both the rate of CO₂ fixation by whole cells and the rate of carboxylation of ribulose-1,5-diphosphate in cell-free extracts are reduced, particularly when sodium acetate is present in the growth medium. Of the enzymes of the reductive pentose phosphate cycle tested, only ribulose-1,5-diphosphate carboxylase activity is reduced in the mutant strain, and it appears that the low carboxylase activity limits the strain's rate of photosynthetic carbon metabolism. Evidence is presented to show that the fluctuation in the level of the enzyme activity in the presence or absence of acetate results from the fluctuation in the level of some factor(s) limiting the rate of synthesis of the protein.     

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.     

A Rapid Method for Secondary-Structure Analysis of the Inulinases of Different Microbial Producers

Computer simulation of secondary structures (calculation of the ratio of α-helices, β-sheets and disordered regions) is a perspective tool needed at the initial stages of the studies of structural and functional features of inulinases, since it enables one to estimate the fluctuation ranges of tested indicators. However, the data from computations should be verified by a number of biophysical and biochemical experimental data, in particular, by experiments using IR-spectroscopy. In the present work, the difference between the experimental and computational data was 3–4% for inulinase from Aspergillus awamori and 12–18% for the enzyme from Kluyveromyces marxianus. Consequently, the analysis of secondary structures of enzymes is applicable for making rapid predictions of the fluctuation ranges of physical?chemical and kinetic characteristics of protein molecules, as well as for rapid evaluation of their dynamic state.     

Analysis of fluctuation in protein abundance without promoter regulation based on Escherichia coli continuous culture

Fluctuation of protein abundance of isogenic Escherichia coli cells in uniform environment was studied. Based on a continuous culture system, which provides homogeneous culture environment, we investigated the fluctuation in GlnA protein abundance regardless of known glnALG promoter regulation. As results by flow cytometer, we found that the GlnA protein abundance in the cells exhibit a large fluctuation, even though GlnA protein is an essential factor for cell growth and the environment is homogeneous. Furthermore, among several steady states, transient processes of such heterogeneous cell population were investigated, by changing the environmental conditions. The results showed that the expression of GlnA protein can be controlled, depending on its necessity, even though there is no known regulatory machinery. These results may provide a clue to understand the nature of regulation of protein expression dynamics with the stochastic fluctuation.     

Analysis of fluctuation in protein abundance without promoter regulation based on Escherichia coli continuous culture

Fluctuation of protein abundance of isogenic Escherichia coli cells in uniform environment was studied. Based on a continuous culture system, which provides homogeneous culture environment, we investigated the fluctuation in GlnA protein abundance regardless of known glnALG promoter regulation. As results by flow cytometer, we found that the GlnA protein abundance in the cells exhibit a large fluctuation, even though GlnA protein is an essential factor for cell growth and the environment is homogeneous. Furthermore, among several steady states, transient processes of such heterogeneous cell population were investigated, by changing the environmental conditions. The results showed that the expression of GlnA protein can be controlled, depending on its necessity, even though there is no known regulatory machinery. These results may provide a clue to understand the nature of regulation of protein expression dynamics with the stochastic fluctuation.     

The coupling of structural fluctuations to hydride transfer in dihydrofolate reductase

The energy barrier for hydride transfer in wild-type G121V and G121S variants of Escherichia coli dihydrofolate reductase (DHFR) fluctuates in a time-dependent manner. This fluctuation may be attributed to structural changes in the protein that modulate the site of chemistry. Despite being far from the active site, mutations at position 121 of DHFR reduce the hydride transfer rate of the enzyme. This occurrence has been suggested to arise from modifications to the conformational ensemble of the protein. We elucidate the effects of the G121S and G121V mutations on the hydride transfer barrier by identifying structural changes in the protein that correlate with lowered barriers. The effect of these structural parameters on the hydride transfer barrier may be rationalized by simple considerations of the geometric constraints of the hydride transfer reaction. Fluctuations of these properties are associated with specific backbone dihedral angles of residues within the Methione-20 (M20) loop. The dihedral angle preferences are mediated by interactions with the region of the enzyme in the vicinity of residue 121 and are translated into distinct ligand conformations. We predict mutations within the M20 loop that may alter the conformational space explored by DHFR. Such mutational changes are anticipated to adjust the hydride transfer efficacy of DHFR by modifying equilibrium distributions of hydride transfer barriers found in the enzyme.     

Distributions of activation energy barriers that produce stretched exponential probability distributions for the time spent in each state of the two state reaction A⇌B

Many biochemical reactions consist of the spontaneous fluctuation between two states: A⇌B. For example these two states could be a ligand bound to an enzyme and the ligand and the enzyme separated from each other. A typical case would be the unbinding of CO from myoglobin (Mb), namely, MbCO⇌Mb+CO. Another example is the fluctuation in the ion channel protein in the cell membrane between conformations that are closed to the passage of ions and those that are open to the passage of ions, namely, closed⇌open. Such chemical reactions can be described as two energy levels corresponding to the two states, separated by a distribution of activation energy barriers. Since a kinetic rate can be associated with each energy barrier, this is also equivalent to a distribution of kinetic rate constants. We derive the distribution of the kinetic rates that produces the stretched exponential probability distribution, exp(−at ^b ) where 0<b≤1, which has been observed for such reactions. We also derive the form of the cumulative probability distribution when the pathways connecting the states have minimum or maximum rate constants.