L-alanine dehydrogenase from Thermus thermophilus.

A heat-stable L-alanine dehydrogenase was isolated and purified from the extremely thermophilic microorganism, Thermus thermophilus, by affinity chromatography. The enzyme has a molecular weight of 290 000, as determined by the sedimentation equilibrium method, and is composed of six subunits of identical molecular weight as concluded from sodium dodecyl sulphate gel electrophoresis. The enzyme has been characterized in terms of pH- and substrate concentration-dependence of activity, substrate specificity, inhibition by D-alanine and D-cysteine and amino acid composition. The parameters obtained are very similar to those reported for L-alanine dehydrogenase from the mesophilic microorganism, Bacillus subtilis (Yoshida, A. and Freese, E. (1965) Biochim. Biophys. Acta 96, 248--262). The thermal stability of the T. thermophilus enzyme is much greater than that of the B. subtilis enzyme. Activation free energy (delta G), activation enthalpy (delta H) and activation entropy (delta S) values were determined for both the alanine deamination and for the heat inactivation reactions of the thermophilic and mesophilic enzymes. The values obtained for the catalytic reaction were practically equal. However, the two enzymes differed significantly in these parameters determined for the enzyme inactivation, which indicates that the factors ensuring the thermoresistance of the enzyme from T. thermophilus do not affect enzyme activity.

Surface hydrophobicity and electrophoretic mobilities of staphylococcal exotoxins with special reference to toxic shock syndrome toxin-1.

The surface hydrophobicities of eleven staphylococcal toxins were estimated and compared with those of standard proteins on an octyl agarose column by high-performance hydrophobic-interaction chromatography (HP-HIC). Staphylococcal enterotoxins (SE) D, C3, C2, C1 and B showed a low surface hydrophobicity whereas alpha-toxin and gamma-toxin had a moderate surface hydrophobicity. SEA, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal epidermolytic toxin (SET) showed high surface hydrophobicity and delta-toxin was the most hydrophobic protein. The electrophoretic mobility of the toxins was determined by free zone electrophoresis (FZE). All toxins except SEC1 and one of the two SEA species showed negative charge at pH 8.6. Charge heterogeneity was observed in SEA, SEC1, SEC3 and TSST-1: SEA and SEC1 had two overlapping components, whereas SEC3 and TSST-1 were resolved into two distinct components. The mobilities of the two TSST-1 components were estimated at -2.12 x 10(-5) and -3.60 x 10(-5) cm2v-1s-1, respectively, at 10 degrees C, and both fractions were immunologically indistinguishable as tested by specific TSST-1 antibodies with ELISA. An asymmetric peak was obtained in hydrophobic-interaction chromatography of TSST-1 indicating heterogeneity.

The mechanism of the inhibitory effect of progesterone on lymphocyte cytotoxicity: I. Progesterone-treated lymphocytes release a substance inhibiting cytotoxicity and prostaglandin synthesis.

Progesterone-treated lymphocytes (generator lymphocytes) of healthy pregnant women release a nondialyzable factor that inhibits both cytotoxic activity and prostaglandin F2 alpha synthesis of test lymphocytes. Production of this factor is blocked by protein synthesis inhibitors (cycloheximide and actinomycin D). Sodium dodecylsulfate polyacrylamide electrophoresis of the partially purified material revealed a main 34,000 MW protein band. Progesterone-treated lymphocytes of pregnant women showing clinical symptoms of threatened preterm delivery (risk group) failed to release this substance.

New set-up for capillary isoelectric focusing in uncoated capillaries.

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis.

Stereoselective interaction of drugs with human serum transferrin in capillary zone electrophoresis is described. The substances passed a pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Drugs with different structures were screened and the enantiomers of bupivacaine, propranolol and promethazine as well as the diastereomers of labetalol were resolved. Racemic mixtures of atenolol and pindolol enantiomers could not be resolved under these conditions.

Unfolding of human serum transferrin in urea studied by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to monitor the progress of the unfolding of human serum transferrin in urea. Denaturation curves of the transferrin forms were constructed plotting the migration times corrected for the viscosity vs. the concentration of urea in the buffer. The practical advantage of capillary zone electrophoresis is the short analysis time, 5-15 min, as compared with slab-gel experiments, which require overnight runs for similar purposes. The resolution increased with the urea concentration, and hence high concentrations are beneficial for quantitative and qualitative analysis of mixtures of transferrin forms. Unfolding intermediates of the isoforms, which interconvert to the unfolded state slowly compared with the time scale of the electrophoretic separation, and also the completely unfolded isoforms were resolved and detected simultaneously when iron-free transferrin was subjected to denaturation by urea at concentrations between 3 and 6 M. However, no unfolding intermediates were observed with transferrin isoforms containing two iron atoms (i.e. diferric transferrin molecules), which accordingly are strongly resistant to urea denaturation. The unfolding of the transferrin isoforms depends on the iron content of the complexes, but not the carbohydrate content. HPCE in the presence of urea in this mode has the potential to become an analytical tool for diagnosis of diseases in which the transferrin patterns change.

Separation of tryptophan-derivative enantiomers with iron-free human serum transferrin by capillary zone electrophoresis.

Enantiomers can be separated by using human serum transferrin as a chiral phase. With the help of the native protein we were able to separate enantiomers with high efficiency, using a low ionic strength 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6, in capillary zone electrophoresis. Tryptophan methyl, ethyl and butyl ester enantiomers-moving towards the cathode at pH 6-were resolved by passing through an iron-free transferrin zone in coated capillaries. Since the isoelectric point of the iron-free transferrin is a little higher than 6, the protein zone is either not moving in the experiment or is slowly moving towards the anode. Under the simplest experimental conditions the highest resolution was obtained for the butyl ester enantiomers and the lowest for the methyl ester ones. By changing the experimental conditions, however, this order could be reversed. The results indicate that the lengths of the alkyl chains in the enantiomers have a significant effect on the resolution, i.e., on the interaction between the protein and the separands.

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.

Separation of the human transferrin isoforms by carrier-free high-performance zone electrophoresis and isoelectric focusing.

Human transferrin isoforms, i.e., molecules with different carbohydrate contents which differ from each other by only one negative charge, were resolved by high-performance zone electrophoresis in free solution. The di-, tri-, tetra-, penta-, hexa- and heptasialo transferrins could be assigned in the electrophoretic pattern. The pattern changed when iron-free transferrin was treated with neuraminidase, which splits off the sialic acid from the carbohydrate chains. The final digest contained transferrin molecules without sialic acids, as was confirmed by isoelectric focusing.

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies.

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.

Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries.

Human serum transferrin is a mixture of isoforms (isoproteins) having different amounts of carbohydrates. Each isoform may exist in iron-free and iron-complexed molecular form. The genetic variations in different populations increase the number of combinations of the different forms of transferrin. To resolve the many components in transferrin preparations, the new high performance capillary technique was employed for isoelectric focusing. Iron-free transferrin and transferrin samples of known iron content were examined. The above method gives an exceptionally rapid analysis (within 15-25 min) of small amounts of samples (less than 1 microgram protein) and as good as or better resolution than other isoelectric focusing techniques previously used for transferrin analysis. By monitoring the focused protein zones at both 280 and 460 nm the molecular forms of transferrin (iron-free, monoferric and differic complexes) can easily be identified. Both steps of isoelectric focusing in capillaries (i.e., prefocusing and mobilization) can be used for analysis. We observed that chelating agents (e.g., carrier ampholytes, nitrilotriacetate) may release iron from microsyringes having metal pistons causing the formation of iron-transferrin complexes.

In situ hybridization histochemistry of mRNAs for hormones and chromogranins in normal pituitary tissue and pituitary adenoma.

In situ hybridization histochemistry was employed to detect mRNAs of pituitary hormones and chromogranins in normal pituitary gland and pituitary adenomas. Oligonucleotide probes specific to the mRNAs for prolactin, growth hormone, proopiomelanocortin, the alpha- and beta-subunits of the glycoprotein hormones and chromogranins A and B were used in the hybridization experiments. The oligonucleotides of 27 to 51 bases were labelled radioactively with dATP[alpha-35S] at the 3'-end using terminal deoxynucleotidyl transferase. Positive hybridization reactions were visualized by autoradiography in the normal pituitary gland with all of the probes. The clinically diagnosed pituitary adenomas (prolactinoma, acromegaly, Cushing's disease, FSH-secreting tumour) showed positive hybridization with the corresponding oligonucleotide probes. In some cases positive hybridization was also obtained with other probes, suggesting multihormone-producing character of the tumour cells. A microprolactinoma was found in a pituitary gland obtained from a patient without any known pituitary disorders. Examination of mRNAs for chromogranin A and B revealed that the normal pituitary gland contains a larger number of cells expressing chromogranin B and a lower number expressing chromogranin A and, moreover, the microprolactinoma lacked the expression of mRNA for chromogranin A but expressed that of chromogranin B.

Influence of thyroid status on postnatal maturation of calcium channels, beta-adrenoceptors and cation transport ATPases in rat ventricular tissue.

In order to examine the influence of thyroid hormones on the postnatal development of cardiac excitation-contraction coupling, newborn rats were made hypo- or hyperthyroid, and several key factors involved, directly or indirectly, in Ca2+ signaling: L-type Ca2+ channels (1,4-dihydropyridine receptors), Ca(2+)-release channels of sarcoplasmic reticulum (ryanodine receptors), beta-adrenoceptors, thapsigargin-sensitive Ca(2+)-ATPase and Na(+)-K(+)-ATPase (enzyme activity and ouabain receptors), were investigated in membrane fractions from ventricular tissue, collected on day 21. Hypothyroidism induced a moderately lower myocardial density of 1,4-dihydropyridine and ryanodinerece receptors (reduced by 23% and 31%, respectively, with respect to euthyroid controls), and much reduced levels of beta-adrenoceptors, Ca(2+)-ATPase and Na(+)-K(+)-ATPase activities. Hyperthyroidism induced only a moderate (22%) decrease in the myocardial density of 1,4-dihydropyridine receptors and a marked (240%) increase of the alpha 2 isoform of Na(+)-K(+)-ATPase. To analyse the subsarcolemmal localization of L-type channels, microsomal fractions were subfractionated by density equilibration in sucrose gradient. In gradients from control and hyperthyroid rats, most 1,4-dihydropyridine receptors were recovered in high-density subfractions, their distribution following that of ryanodine receptors, whereas, in gradients from hypothyroid rats, most 1,4-dihydropyridine receptors were recovered in low-density subfractions, together with beta-adrenoceptors and Na(+)-K(+)-ATPase. We conclude that thyroid hormones are important for the postnatal changes in the myocardial density of several channels and pumps involved in Ca2+ fluxes, as well as for the postnatal redistribution of L-type Ca2+ channels from non-junctional sarcolemma to junctional structures, a key process for the efficient operation of excitation-contraction coupling in adult ventricular tissue.

Stereoselective interaction of drug enantiomers with human serum transferrin in capillary zone electrophoresis (II).

Further studies on chiral resolution of drugs with different chemical structures by capillary zone electrophoresis using iron-free human serum transferrin are described. The substances passed a highly concentrated pseudo-stationary protein zone applied in a coated capillary and the possible chiral separation of the optical isomers was followed. Eighteen drugs with different structures were screened, and the enantiomers of clofedanol, buphenine, acebutolol and chlorphenamine were resolved. Several, but not all drugs, showed longer migration times while passing the protein zone, indicating an interaction with transferrin, although chiral resolution was not observed in all cases. The observations provided further information about the properties of the surface interaction sites of transferrin.

Different segmental flexibility of human serum transferrin and lactoferrin.

X-ray diffraction studies show that the diferric (holo) forms of human serum transferrin and lactoferrin have almost the same conformation in crystal. In solution, however, the two proteins exhibit different characteristics. The differences are even more pronounced in the apo forms. Small-angle X-ray and neutron scattering data show that lactoferrin is less compact, in apo and holo forms, than the corresponding forms of transferrin in solution. The comparison of primary structures of the two proteins suggests that one of the interdomain hinge regions is significantly longer in lactoferrin than its counterpart in transferrin. The difference in flexibility due to the long hinge region in lactoferrin may be responsible for many of the differences in the physicochemical characteristics of the two proteins.

Zero-field Mössbauer studies of diferric human transferrin.

Diferric transferrin samples labelled with 57Fe at the N- or the C-terminal binding sites are compared by Mössbauer spectroscopy at 15 K and in zero magnetic field. The spectra of the samples are similar but the fitting of single Lorenzian lines to the data shows that some of the line positions differ in the two cases. According to this we can not exclude a difference between the chemical structures of the binding sites that can arise for example from the participation of different forms of the anion and/or water in the two lobes of transferrin. All other line parameters (line-width, intensity) are the same within the limits of errors.

Chiral separation of alpha-amino acids by ligand-exchange capillary electrophoresis using N-(2-hydroxy-octyl)-L-4-hydroxyproline as a selector.

The direct chiral resolution of underivatized alpha-amino acids by capillary zone electrophoresis (CZE) based on the principle of ligand exchange is described. An N-(2-hydroxyoctyl)-L-4-hydroxyproline/Cu(II) complex was used as a chiral selector. Besides amino acids containing aromatic residues, the basic amino acid histidine was resolved. Baseline separations were obtained for all amino acids investigated. The influence of selector concentration, electrolyte composition and pH on the resolution was investigated. It was found that there is a correlation between pI of the amino acids and the optimal pH.

Protein profile characterization of bacterial lysates by capillary electrophoresis.

A fast and reproducible method was developed to characterize cell lysates by their electrophoretic profiles using capillary electrophoresis (CE). Characteristic and reproducible patterns were recorded for each bacterial strains when "dynamic sieving" CE, using a polymer solution in the capillary, was applied to distinguish four strains of the Enterobacteriaceae family. The electropherograms showed distinct differences when comparing them to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. This is certainly a result of the differences in the separation principles and in the detection methods of the two techniques.