Shunji Nagaoka

High-performance liquid chromatographic studies on non-mercapt α mercapt conversion of human serum albumin. II

Abstract High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on a GS-520 column with 0.03 M sodium phosphate buffer—0.15 M sodium sulphate (pH 6.87) showed three peaks, the principal component corresponding to human mercaptalbumin (HMA) and the secondary and tertiary components to nonmercaptalbumin (HNA). Using HPLC analysis, the nonmercapt → mercapt conversion of HSA during haemodialysis and the mercapt → nonmercapt conversion after haemodialysis in chronic renal failure were re-confirmed, indicating that HMA is a covalent carrier protein for sulphur-containing amino acids. Fractions of HMA in various liver diseases were significantly lower than those of healthy male adults.

Heat denaturation of bovine serum albumin in alkaline pH region

Abstract The heat denaturation of bovine serum albumin was studied at 65°C in 0.1 M Tris-EDTA-boric acid buffer at pH 9.0. The gel electrophoresis indicated that the heat-treated bovine serum albumin solution contained several components, 1, 1′, 2, 3 and others. It was concluded that the component 1′ is a monomeric bovine serum albumin modified by the intramolecular SH-S-S exchange reaction. When the SH-blocked bovine serum albumin was heated in the presence of cysteine, a component having the same mobility as the component 1′, was formed by the reaction between the SH group of cysteine and the SS bond in bovine serum albumin, or by the SH-catalyzed structural alteration. (Components 1, 2 and 3 are a monomer, dimer and probably trimer of bovine serum albumin, respectively, as has already been concluded.) The amount of 1′ depended on the concentration of Tris-EDTA-boric acid buffer. The higher concentration of the buffer favored the formation of 1′, i.e. the intramolecular reaction. Components 1, 1′, 2 and 3 were isolated by the preparative disc gel electrophoresis. Studies on each component revealed the following: (a) The extinction coefficient E1 cm1% was the same for components 1, 1′, 2 and 3, being 6.6–6.7 at 279 nm, (b) The sedimentation coefficient of 1 and 1′ differed slightly, and (c) The helical content decreased in the order 1 > 1′ > 2 > 3. An evidence to support the concept of microheterogeneity of bovine serum albumin was presented. Additionally, the alkali denaturation of bovine serum albumin was studied in the pH range 10.5–12.0.

Fluorimetric studies on the structural transition of bovine plasma albumin in acidic solutions

The acid-induced isomerization (the NF transition) and expansion of bovine plasma albumin were studied by measuring [α]233 and tryptophyl fluorescence. Most of the changes in tryptophyl fluorescence intensity at 350 nm correlated exactly with the NF1 transition and/or the initial part of the NF transition.

Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artiifical membrane systems

Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids.

Comparative 1H-NMR studies on the physical state of water in soft contact lens and mouse lens

The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin-lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%.

Dimerization of bovine plasma albumin in low pH region

Abstract The dimerization of charcoal-defatted and conventionally defatted bovine plasma albumin in 0.10 M KCl at low pH was re-examined using a sedimentation velocity method. The results obtained did not substantiate the previous conclusion that dimerization was significant near pH 3.3. The addition of Cu 2+ caused significant dimerization of bovine plasma albumin in 0.10 M KCl at pH 3.30.

Conformational change of bovine serum albumin induced by 0.10 M Tris-EDTA-boric acid buffer at pH 9

Abstract The sedimentation coefficient of bovine serum albumin was 4.40 S in 0.02 M Tris-EDTA-boric acid buffer, while it was 3.95 S in the same buffer at 0.10 M. It is concluded that the 0.10 M Tris-EDTA-boric acid buffer at pH 9.0 induces a conformational change in bovine serum albumin. Data of optical rotatory dispersion and of −[α]233 support this conclusion.

PHOSPHATIDYL INOSITOL AS A BINDING COMPONENT OF OPIOID RECEPTOR

A direct approach to the interaction between opioids and phosphatidyl inositol (PI)-liposome was studied by a fluoropolarimetric method to examine a rotational freedom of bound opioids using the intrinsic fluorescence of the compounds. PI storongly immobilized the rotational motion of the agonist molecule (etorphine, morphine and levorphanol) and the partial antagonist (levallorphan and pentazocine). This PI-opioid interaction decreased in the presence of calcium ion. On the contrary, local anesthetics, such as procaine or tetracaine, were immobilized by PI and other lipid system even in the presence of calcium ion. 3-Methyl derivative of levorphanol showed little interaction with PI. Dextrorphan also interacted with PI to some degrees. β-Endorphin was immobilized slightly by binding to PI, but enkephalines and kyotorphin were not immobilized. These results are conceivable that PI is an important component for the binding site of μ-type opioid receptor.

A ROLE OF LIPID IN OPIATE-RECEPTOR INTERACTION

The interaction between opiates and lipids vesicle was studied by fluoripolarimetric method to examine a rotational freedom of bound opiates using the intrinsec fluorescence of opiates. Among the selected combinations of the lipid-opiate system tested, phosphatidyl inositol showed the highest affinity to morphine and its analogues, resulting in a strong immobilization of bound opiates, whereas the other phospholipids and cerebroside sulfate did not alter the dynamic property of the opiates even certain interaction was expected from the change in fluorescence intensity. Phosphatidyl serine which quenched the opiate fluorescence, also showed a slight immobilization of some opiates. On the contrary, the endogenous opioids were not immobilized even by phosphatidyl inositol.