Studies on yellow fever vaccine. II--Stability of the reconstituted product

This work evaluated the stability of diluted yellow fever vaccine in order to determine conditions that maintain the minimum of 3 log10 of 17D virus per human dose as required by WHO. The vaccines were held at 0 degrees C or at 37 degrees C and were diluted either with distilled water, with 0.15 M saline or with 0.15 M PBS at pH 5.5, 7.2 and 8.0. In a next step, stabilizer substances such as gelatin and peptone were added to the vaccines. Dilution of the vaccines in distilled water maintained the virus titre for up to three hours at 37 degrees C and this diluent has been adopted for routine use in Brazil.     

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.     

Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli

Solubilized glycerophosphate acyltransferase from Escherichia coli was reconstituted in small unilamellar vesicles consisting of phosphatidylcholine/phosphatidylglycerol in a molar ratio of 4:1. Glycerol 3-phosphate, trapped inside these vesicles, cannot be acylated by the enzyme upon addition of extra-vesicular palmitoyl-CoA. Thus, substrate-binding sites and active sites are asymmetrically oriented in the model membrane. When up to 10 mol/100 mol lysophosphatidic acid was incorporated in the vesicles a decrease in glycerophosphate acyltransferase activity is observed at amounts exceeding 1 mol% lysophosphatidate. Similar experiments, using lysophosphatidylcholine and phosphatidic acid, suggest the decrease to result from an increase in negative surface charge. Reconstituted glycerophosphate acyltransferase exhibits a preference for palmitoyl-CoA over oleoyl-CoA. This preference increases considerably at elevated temperatures. The glycerophosphate acyltransferase could, therefore, participate in the temperature-dependent changes in the fatty acid composition of the phospholipids in E. coli.     

Studies on a reconstituted acetylcholine receptor system: effect of agonists

An impure preparation of acetylcholinesterase from electroplax of the electric eel can be incorporated into a bimolecular lipid membrane. The acetylcholinesterase-modified bimolecular lipid membrane shows a concentration-dependent increase in membrane conductance elicited by several agonists (acetylcholine, carbamylcholine, phenyltrimethylammonium ion, tetraethylammonium ion, decamethonium ion, and nicotine) added to the compartment opposite that to which acetylcholinesterase was originally added. Affinity and efficacy of the various agonists in generating the conductance increase were measured from dose-response curves; these are in good quantitative agreement with corresponding values observed for depolarization of intact eel electroplax. The ion conduction pathways induced by agonists in the modified bimolecular lipid membrane show a slight cation selectivity, Na ? K > Cl (3:3:1), similar to that observed for the depolarized electroplax membrane. Evidence is presented that suggests that some components other than acetylcholinesterase induce the acetylcholine receptor response in the bimolecular lipid membrane.     

Spectral properties of perselenosulfide

The riductive scission of a diselenide (selenocystamine) produced by disulfide gives the perselenosulfide, a new compound. Its formation kinetics, carried out a several pH values, were recorded spectrophotometrically at 375 nm. The lability of perselenosulfide bond by cianolysis has been demonstrated.     

Influence of C-protein on mechano-chemical properties and structure of reconstituted actomyosin

C-protein on the mechano-chemical properties (ATPase activity and superprecipitation) of actomyosin systems has been investigated. The presence of C-protein in AM-complexes has been shown to decrease the rate of superprecipitation (SPP) and simultaneously increase the ATPase activity. Both effects of C-protein are dependent on its quantity in the system. Tropomyosin decreased considerably but does not eliminate completely the inhibitory influence of C-protein on the SPP. Electron microscopy does not reveal considerable structural differences in the initial AM-complexes depending on the presence or absence of C-protein. It is supposed that the discovered effects of C-protein on the behaviour of AM-systems are determined by the fine local structural and (or) charge changes produced by C-protein in the region of myosin cross-bridges, which in its turn results in a modification of the actin-myosin interaction. Possible participation of C-protein in the regulation of the interaction of thin and thick filaments in the muscle is discussed.     

胆碱脱氢酶光谱性质的研究

胆碱脱氢酶（ＣＤＨ）蛋白质部分内源荧光发射峰在３３５ｎｍ,并不受底物的影响,但底物可改变辅基ＦＡＤ部分的内源荧光光谱。应用ＦＴＩＲ技术研究了增溶ＣＤＨ的二级结构,其结果如下：５３．４％α－螺旋,２４．５％β－片层,１３．９％３１０－螺旋及０．５％β－回折。在ＣＤＨ处于非底物结合状态时,分子内部结构表现为α－螺旋以及β－片层优势构象,呈现出球状蛋白样的空间结构特征。在与底物作用过程中,３１０－螺旋的比例逐渐上升至４２％左右,与此同时α－螺旋结构则降低到３５％。提示了底物诱导ＣＤＨ分子内部发生了蛋白分子的重新折叠。     

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.     

Studies on the hydrophobic properties of sphingomyelinase.

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.     

Studies on the neurochemical properties of cystathionine.

Following the intracerebral administration of [35S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus. The uptake of [35S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters. Depolarization of the synaptic membrane had no effect on the efflux of [35S]cystathionine from preloaded synaptosomes. The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.     

Catalytic properties of the liver microsomal hydroxylase system in reconstituted phospholipid vesicles.

Two types of cytochrome P-450, P-450LM2 and P-450LM3, have been purified from rabbit liver microsomes and incorporated into phospholipid vesicles by a cholate gel filtration technique together with purified preparations of NADPH-cytochrome P-450 reductase. The catalytic properties of the vesicles have been compared with a system reconstituted with small amounts of dilauroylphosphatidylcholine (DLPC). 6 beta-Hydroxylation of androstenedione proceeded at a rate 10 times higher in the vesicles compared to the DLPC-system. The kinetics for the reaction were the same in the vesicles as in intact microsomes i.e. sigmoidal substrate curves were obtained and Hill-coefficients of about 1.4 were calculated in these systems. In contrast, Michaelis-Menten kinetics were obtained for 6 beta-hydroxylation in the DLPC-system. The results could indicate cooperativity between different P-450 molecules in the intact membrane but not in the DLPC-system. P-450LM2-catalyzed 16-hydroxylation of androstenedione was in contrast to the situation with P-450LM3 inhibited in the vesicles as compared to the DLPC system. It is suggested that for evaluation of substrate specificity and other properties of different types of liver microsomal P-450, phospholipid vesicles may be a more relevant integration level than the DLPC-system.     

Influence of telopeptide on the morphology and physicomechanical properties of reconstituted collagens

The morphology and physicomechanical properties of two types of collagen membranes, one of which does not have telopeptides, were compared with small-angle light scattering, rheo-optical, dynamic mechanical, and dynamic rheo-optical techniques. The presence of telopeptides in native collagen allows the formation of larger rod-like superstructures; it renders the membrane more resistant to irreversible deformation yet more responsive to dynamic mechanical perturbation. Telopeptides are also responsible for relaxation at room temperature, and impart more linearly elastic properties to the material as compared to membranes derived from enzyme-treated collagens.     

Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase

Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze-fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity.     

Effects of local anesthetics on properties of tetrodotoxin-sensitive protein reconstituted into liposomes

The effects of some local anesthetics on properties of tetrodotoxin (TTX)-sensitive protein reconstituted into liposomes in such a manner that its TTX-sensitive center is located at the internal surface of the liposome membrane were studied. It was shown that tetracaine, lidocaine and its derivative QX-314 decreased the rate of efflux of radioactive sodium from the²²Na-preloaded proteoliposomes with the same efficiency as TTX acted from the inside of liposomes. The results confirm our earlier suggestion that TTX-sensitive protein is a soluble precursor of the protein forming voltage-dependent sodium channels.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 299–302, July–August, 1995.