The complete amino-acid sequence of the bilin-binding protein from Pieris brassicae and its similarity to a family of serum transport proteins like the retinol-binding proteins

The amino-acid sequence from the bilin binding protein (BBP) of the butterfly Pieris brassicae has been determined. The apoprotein with a length of 173 amino-acid residues has a molecular mass of 19,676 Da. The sequence analysis was performed by automated Edman degradation of the intact apoprotein and of fragments as large as possible generated from different digestions. The 3-dimensional structure of BBP, determined by Huber et al. (Huber, R., Schneider, M., Epp, O., Mayr, I., Messerschmidt, A., Pflugrath, J. & Kayser, H. (1987) J. Mol. Biol. 195, 423-434 and Huber, R., Schneider, M., Mayr, I., Müller, R., Deutzmann, R., Suter, F., Zuber, H., Falk, H. & Kayser, H. (1987) J. Mol. Biol. 198, 499-513) down to 2-A resolution, exhibits a similar conformation to the human retinol binding protein. Sawyer (Sawyer, L. (1987) Nature (London) 327, 659) demonstrated that proteins from a wide variety of sources can be gathered into a "superfamily". Computer searches of data banks yielded in a new member of this superfamily, namely human alpha 1-acid glycoprotein. One of the functions of the listed proteins is to bind and transport small hydrophobic molecules in serum.     

Versatility of anti-horseradish peroxidase antibody-gold complexes for cytochemistry and in situ hybridization: preparation and application of soluble complexes with streptavidin-peroxidase conjugates and biotinylated antibodies

Summary In previous studies we have employed a gold-labelled, affinity-purified polyclonal antibody against horseradish peroxidase (anti-HRP — gold) in the avidinbiotin peroxidase complex (ABC) technique and indirect labelled avidin-biotin methods. The gold-labelled antibody was used as final revealing reagent to replace the 3,3-diaminobenzidine (DAB) reaction by immunogold silver staining. The anti-HRP — gold reagent proved to be advantageous since blocking of endogenous peroxidase activity in the tissue sections was not further required and staining of superior contrast and resolution could be achieved in paraffin sections. In the present study we have optimized this technique by combining the last two incubation steps, i.e. HRP-conjugated streptavidin and anti-HRP — gold. Different ratios of the two reagents were tested empirically to establish the conditions for the formation of a soluble complex with optimal staining properties. Quantitative evaluation by densitometry of the staining intensity showed that the soluble streptavidin-HRP/anti-HRP — gold complex and the indirect labelled avidin-biotin method employing the gold-labelled anti-HRP antibody performed equally well. Thus, the availability of this complex simplifies the streptavidin-biotin immunogold technique for immunohistochemistry, lectin histochemistry and in situ hybridization and further demonstrates the versatility of anti-HRP — gold complexes.     

Expression of polysialylated N-CAM during rat heart development

Developmental patterns of immunoreactivity for the neural cell adhesion molecule (N-CAM) and alpha 2.8-linked polysialic acid (PSA) were identified in embryonic and postnatal rat heart by immunocytochemistry and immunoblotting. Polyclonal antibodies against N-CAM and a monoclonal antibody which recognises only polymers of PSA with a chain length greater than eight units were used. Gold- and alkaline-phosphatase-labelled antibodies were used for detection. The N-CAM polypeptide isoform pattern seen by immunoblotting after endoneuraminidase treatment changed as development progressed. During embryonic development a 160-kDa polypeptide isoform was predominant. Around birth, 130-, 160- and 170-kDa polypeptide isoforms were found. The expression of the 130- and 170-kDa isoforms diminished until finally, in the adult, weak immunoreactivity for bands of 120-, 130- and 160-kDa was seen. In general the extent and intensity of PSA and N-CAM immunostaining in rat heart increased until birth and declined thereafter. Early in development prominent immunostaining for PSA and N-CAM was seen in the epicardium while later in development this area was only weakly stained. Initially myocardial cells, endocardial cells and some cells in the atrioventricular cushions were immunoreactive for both PSA and N-CAM. Later in development N-CAM immunostaining was more prominent than PSA immunoreactivity, reflecting a decrease in N-CAM polysialylation, which was also seen by immunoblotting. During innervation of the heart, nerve fibres were strongly immunostained for PSA and N-CAM, and this was the only immunostaining seen in adult heart.     

A smooth muscle cell line suitable for the study of voltage sensitive calcium channels

A cell line originating from the fetal rat aorta has been studied with respect to 45Ca2+ uptake. Kinetic experiments showed an initial rapid uptake followed by a slow linear phase; both the initial rate and the maximum uptake were increased in the presence of 55 mM potassium chloride. The calcium channel antagonists, darodipine (PY 108-068) and verapamil, inhibited both the basal and the potassium chloride stimulated uptake. Neither tetrodotoxin nor furosemide affected either basal or depolarisation induced 45Ca2+ uptake. Blockade of the Na+/K+ ATPase by ouabain and of the Ca2+ ATPase by vanadate caused a net increase in cellular 45Ca2+ accumulation.     

Elevation of the number of cell-surface insulin receptors and the rate of 2-deoxyglucose uptake by exposure of 3T3-L1 adipocytes to tolbutamide

Sulfonylurea compounds are hypoglycemic agents which by unknown mechanisms alter the amount of insulin receptor and the rate of glucose utilization in tissues exposed to the drugs. In this study the effects on insulin binding and uptake of 2-deoxyglucose by 3T3-L1 adipocytes were assessed after maintaining cell monolayers for 1-3 days in medium containing different concentrations of the sulfonylurea, tolbutamide. The amount of 125I-insulin bound by treated monolayers gradually increased to values 150-250% of those of control monolayers after 2-3 days of exposure to 1.5 mM tolbutamide. Such increases in insulin binding capacity arose primarily from an increase in receptor number and not from an alteration in the affinity of the receptor for insulin. Concomitant with the changes observed for the insulin receptor, tolbutamide-treated monolayers expressed 1.5-2-fold higher rates of uptake of 2-deoxyglucose relative to control monolayers at concentrations of insulin between 0 and 10(-10) M. This study thus demonstrates the responsiveness of adipocytes to tolbutamide and also establishes the usefulness of 3T3-L1 cells as a model system in which to study the mechanism of tolbutamide action, both as it relates to the use of sulfonylurea compounds in clinical applications and as possible probes for perturbing and studying relatively uncharacterized regulatory pathways controlling receptor level and biological responses to insulin.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

Frequency characteristics of the saccadic eye movement

Using a piecewise linear approach, individual saccadic eye movements have been Fourier decomposed in an attempt to determine the effect of saccadic amplitude on frequency characteristics. These characteristics were plotted in the traditional Bode plot form, showing gain and phase as a function of frequency for various eye movement amplitudes. Up to about one octave beyond the -3 db gain frequency, the limiting system dynamics represented by the saccadic trajectory of a given amplitude may be considered linear and second order. The -3 db gain frequency was used as a measure of bandwidth, and the -90 degrees phase crossover frequency was used as a measure of undamped natural frequency. These two quantities were used to calculate the damping factor. Both bandwidth and undamped natural frequency decrease with increasing saccadic eye movement amplitude. The damping factor shows no trend with amplitude and indicates approximate critical damping. When compared with the normal variation of characteristics for a given movement, the frequency characteristics of fixed-amplitude saccades showed no generalized trends with changes in direction or DC operating level of movement.     

Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A

The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 A resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism. Hexagonal phycoerythrocyanin crystals of space group P6(3) with cell constants a = b = 156.86 A, c = 40.39 A, alpha = beta = 90 degrees, gamma = 120 degrees are almost isomorphous to C-phycocyanin crystals. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 A resolution. In phycoerythrocyanin, the three (alpha beta)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin. The two structures are very similar. After superposition, the 162 C alpha atoms of the alpha-subunit have a mean difference of 0.71 A and the 171 C alpha atoms of the beta-subunit differ by 0.51 A. The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C(31)-R, C(4)-S. The configuration of the chromophore is C(10)-Z, C(15)-Z and the conformation C(5)-anti, C(9)-syn and C(14)-anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.     

Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.