Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum.

1. Sinapis alba L. seedlings contain glycosyltransferase catalyzing the synthesis of sterol glucosides in the presence of UDPglucose as sugar donor. The major activity occurs in the membranous fraction sedimenting at 300--9000 x g. Successive treatment of the particulate enzyme fraction with acetone and Triton X-100 affords a soluble glucosyltransferase preparation which can be partly purified by gel filtration on Sephadex G-150. Molecular weight of the glucosyltransferase is 1.4 . 10(5). Apparent Km values for UDPglucose and sitosterol are 8.0 . 10(-5) M and 5.0 . 10(-6) M, respectively. 2. Comparison was made of the S. alba glucosyltransferase with a similar sterol-glucosylating enzyme isolated from non-photosynthesizing organism Physarum polycephalum (Myxomycetes). UDPglucose was the most efficient glucose donor in both cases but the enzyme from Ph. polycephalum can also utilize CDPglucose and TDPglucose. Glucose acceptors are, in case of both enzymes, sterols containing a beta-OH group at C-3 and a planar ring system (5 alpha-H or double bond at C-5). The number and position of double bonds in the ring system and in the side chain, as well as the presence of additional alkyl groups in the side chain at C-24 are of secondary importance. 3. The present results indicate that both enzymes can be regarded as specific UDPglucose:sterol glucosyltransferases. Certain differences in their specificity towards donors and acceptors of the glucosyl moiety suggest, however, a different structure of the active sites in both enzymes.     

Studies on the reaction between human growth hormone and oleic acid using polyacrylamide gel electrophoresis.

Through the work of U. J. Lewis and E. V. Cheever (1967, Endocrinoloyg81, 1338–1348) and U. J. Lewis, E. V. Cheever, and B. K. Seavey (1968, J. Biol. Chem.243, 260–267) it has been known for a number of years that human growth hormone (hGH), and many other proteins, reacts with unsaturated fatty acids to give rise to species with enhanced electrophoretic mobility. In view of the possible importance of this reaction in the genesis of charge isomeric protein artifacts, and for the understanding of hGH as a system of multiple isomers with distinct, and in some cases enhanced, specific activities, the nature of this reaction was investigated further. It was found that (1) the positions of oleic acid and growth hormone on polyacrylamide gel electrophoresis (PAGE) are coincident, indicating that the reaction leads to binding of the fatty acid to the protein: (2) the increment in molecular net charge on growth hormone is proportional to the molar ratio between the reactants, oleic acid and hGH; (3) the binding is noncovalent since it reverses under conditions of isoelectric focusing; (4) the reaction product has a molecular size indistinguishable from that of the reactant, hGH, by the criteria of “quantitative” PAGE (however, the reaction product exhibits an elevated negative molecular net charge in PAGE at pH 10.2); (5) the apparent isoelectric points of the hormone and its reaction products with oleic acid are indistinguishable in isoelectric focusing; (6) the interaction does not seem to involve the carboxyl charges on oleic acid since it is independent of ionic strength; (7) a noncovalent hydrophobic interaction with the protein is indicated since the range of electrophoretic mobilities exhibited by the hGH-oleic acid complex is smaller in the presence of benzyl alcohol in the gel than that exhibited by controls in it absence; (8) the reaction does not seem to involve free radical derivatives of the unsaturated fatty acid since it is not altered when the polyacrylamide gel is in a nonoxidative state; (9) the effect of the reaction with oleic acid on the tryptophan spectrum reflects only nonspecific interaction of the hormone-concomitant tryptophan perturbation.     

Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.     

Untangling spider silk evolution with spidroin terminal domains

Background  

Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains.     

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.     

Thermoregulation during prolonged exercise in heat: alterations with beta-adrenergic blockade

Thermoregulation and cardiovascular drift were studied under conditions of prolonged exercise in a warm environment (dry bulb temperature 31.7 +/- 0.3 degrees C, rh 44.7 +/- 4.7%) during beta-adrenergic blockade. Fourteen subjects performed 90-min rides on a cycle ergometer at a work rate equivalent to 40% of their control maximal O2 uptake under each of three treatments provided in a randomized double-blind manner: atenolol (100 mg/day), propranolol (160 mg/day), and a placebo. Exercise during the propranolol trial resulted in significantly higher forearm vascular resistance values and significantly lower forearm blood flows (FBF) compared with the placebo trial. However, the significantly lower FBF during propranolol did not significantly alter the rectal temperature (Tre) response to prolonged exercise. In addition, both beta-blockers produced lower FBF for any given Tre, suggesting that beta-adrenergic blockade affects FBF through nonthermal factors. The slight differences in Tre, despite the large differences in FBF between the various treatments, are apparently the result of an enhanced sweat loss and a lower mean skin temperature during exercise with beta-blockade. The uncoupling of FBF and sweat loss provides evidence of independent regulation. The reduction in FBF at any given Tre was concomitant to lower blood pressure values during beta-blockade and suggests that baroreflexes provide significant input to the control of skin blood flow when both pressure and temperature maintenance are simultaneously challenged.     

In vitro assessment of the selectivities of various beta-adrenergic blocking agents

Intrinsic sympathomimetic- and membrane activities of beta-adrenoceptor blocking agents are of little or no clinical significance. A selective blockade of cardiac receptors has important therapeutic consequences, especially in the treatment of patients with obstructive airways diseases. Profound depression of miocardial contractility can be deleterious in patients with cardiac muscle damage and the use of beta-adrenergic blockers with a quantitative selectivity towards chronotropism may become an important consideration. The effects of a number of beta-sympatholytics have been determined on isolated cardiac preparations (beta₁-adrenergic receptors) and tracheal preparations (beta₂-adrenergic receptors) of guinea-pigs. Results indicate that prindolol is the most selective blocker of the beta₁ chronotropic receptors whilst atenolol could be classified as being the most cardioselective beta-adrenergic blocker investigated. Butoxamine, on the other hand, proved to be the most beta₂-selective one.     

UVB radiation induced effects on cells studied by FTIR spectroscopy

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm²) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spetroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698–705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.