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Biomedical subjects

A Ikai

Publications and source records attributed to A Ikai.

143 records · Page 8Linked to original sources
PubMed1977

Proteolysis of apoproteins in human serum low density lipoprotein.

Proteolytic treatment of human serum low density lipoprotein (LDL) resulted in the observation of interesting time-dependent changes in the sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of apo-LDL. Five major fragments with well-defined relative mobilities appeared within 30 min of protease treatment. Prolonged treatment with subtilisin caused changes in the amount of peptides in each of the five bands but their positions on the gel remained unchanged. Periodic acid-Schiff base staining of the gel showed a proteolytic fragment with an apparent molecular weight of 110.000 (actually a cross-linked dimer of two peptides with molecular weights of 77,000 and 68,000) to be a carbohydrate-bearing peptide that was most resistant to further proteolysis and therefore responsible for the interaction between the digested LDL and concanavalin A.

Apolipoproteins↗
PubMed1977

Properties of opsin-lipid complex in aqueous 2-chloroethanol.

Cattle and squid opsins were found to be associated with phospholipids after extensive dialysis of the salt-free digitonin extract of rhodopsin against 30% aqueous 2-chloroethanol (v/v) at pH 2.5. The approximates sizes of opsin-lipid complexes were estimated by sedimentation studies to be around 110,000 and 150,000 daltons, respectively, for the cattle and the squid opsin. Phospholipids did not dissociate from opsin even in 80% 2-chloroethanol. The complexes were purified by passage through a Sephadex G-200 gel column equilibrated with 30% 2-chloroethanol. The optical properties of the complex suggested the presence of beta-conformation and a small amount of alpha-helix in solubilized cattle opsin.

Animals↗
PubMed1976

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.

Guanidines↗
PubMed1976

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.

Binding Sites↗
PubMed1976

Stepwise degradation of serum low denisty lipoprotein by sodium dodecyl sulfate.

The structure of human serum low density lipoprotein (LDL) was investigated by perturbing the LDL structure with sodium dodecyl sulfate (SDS). The change in LDL structure induced by the addition of SDS was monitored by sedimentation velocity measurements, ultraviolet difference spectroscopy, fluorescence spectroscopy and proteolytic digestion of apo-LDL with subtilisin BPN' [EC 3.4.21.14]. As the concentration of SDS was increased from 0.1 mg/ml to 3 mg/ml with LDL concentrations between 2.0 mg/ml and 4.4 mg/ml, the sedimentation coefficient of LDL changed in three distinct steps. It was found by chemical analyses that not more than 30% of the total lipid was lost from LDL in the second step, whereas the final step in the change of sedimentation coefficient corresponded to the complete removal of apo-LDL from the constituent lipids of LDL. The ultraviolet difference spectrum between the native and SDS-treated LDL and the quenching of LDL fluorescence underwent about 80% of the total change while the SDS concentration was only sufficient to cause the second of the three step changes in sedimentation coefficient. SDS-polyacrylamide gel electrophoresis of apo-LDL treated with subtilisin BPN' also showed that more than 70% of apo-LDL became susceptible to proteolysis under the same conditions. These results were interpreted as indicating that the solubilization of 20 to 30% of the lipids on the surface of LDL exposed nearly 80% or more of apo-LDL to the solvent. A small portion of apo-LDL was, however, still firmly anchored to the remaining lipid micelle as long as the concentration of SDS was less than that required to cause the final step of the change in sedimentation coefficient.

Animals↗
PubMed1975

Denaturation and proteolytic digestion of porcine low-density lipoprotein in aqueous guanidine hydrochloride solutions.

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.

Animals↗
PubMedDate not supplied

Molecular characterization of the "26S" proteasome complex from rat liver.

The molecular properties of an ATP/ubiquitin-dependent "26S" proteasome complex purified from rat liver were examined by physicochemical, biochemical, and morphological analyses. On ultracentrifugation, the proteasome complex sedimented as almost a single component with a sedimentation coefficient of 30.3S. Dynamic light-scattering measurements indicated that it has a diffusion coefficient of 1.38 x 10(-7) cm2/sec and a Stokes radius of 15.5 nm. From these two coefficients, the protein complex was estimated to have the high molecular weight of 2.02 x 10(6). Static light-scattering analysis indicated a molecular weight of 1.91 x 10(6) and a radius of gyration of 16.8 nm. The proteasome complex was found to be composed of multiple subunits of the 20S proteasome with molecular weights of 2.1-3.1 x 10(4) and 15-20 protein species with molecular weights of 3.5-11.0 x 10(4), which were directly associated with the 20S proteasome. The electron micrographic finding that the 26S proteasome complex had a caterpillar shape, direct electronmicroscopic observations on the subunit arrangement of the 20S proteasome, and classification of the subunits of the latter into two groups with respect to sequence homology suggested that the 26S complex is a symmetrical assembly of two domains, each containing a large terminal subset and half the central 20S subset of components. For clarification of the molecular structure of the 26S proteasome complex in solution, its physicochemical parameters were calculated theoretically using a model based on this caterpillar-shaped complex. The values obtained for the Stokes radius and radius of gyration of 12.2 and 14.9 nm were consistent with the experimental values. These results provide evidence that the 26S proteasome complex is a cylindrical caterpillar-like structure of "30S" in solution, consisting of a 20S proteasome component with proteolytic function and multiple other components, which possibly have regulatory roles.

Adenosine Triphosphate↗
PubMedDate not supplied

Open quaternary structure of the hagfish proteinase inhibitor with similar properties to human alpha-2-macroglobulin.

A homologous protein to human plasma alpha-2-macroglobulin (alpha-2-M) was purified from the blood plasma of hagfish (Eptatretus buergeri) and its structure and function were studied. The hagfish protein inhibited several proteinases and its inhibitory activity was blocked with methylamine as in the case of human alpha-2-M. The molecular weight and sedimentation coefficient of the hagfish inhibitor were 390,000 +/- 20,000 and 11.0 S, respectively, as determined by sedimentation studies. The frictional ratio calculated from these parameters was 1.75. The Stokes radius estimated from HPLC gel chromatography was 8.8-8.9 nm, which was similar to that of human alpha-2-M despite the fact that the hagfish inhibitor was only one-half as large as human alpha-2-M in molecular weight. The hagfish inhibitor was expected to be more asymmetric and/or more hydrated than the human inhibitor. The electron micrographs of the negatively stained hagfish inhibitor showed that it had an open, rectangular quaternary structure of 15 +/- 1.5 X 19 +/- 2 nm in which two semiglobular units were located at the two shorter sides with a gap of 8 +/- 1 nm in width. Each semiglobular unit had an approximate width of 5 +/- 0.5 nm. The thickness of the unit was estimated to be 3 to 3.5 nm from the result of fixed-angle shadowing experiments. Although the two semiglobular units must be connected by some structure, very little material could be seen between them. Such an open quaternary structure may explain the high frictional ratio and large Stokes radius of this protein. The structural change of the inhibitor after reaction with proteinases or methylamine could be detected by electron microscopy and gel chromatography.

Amino Acids↗
PubMedDate not supplied

Calculation and experimental verification of the frictional ratio of hagfish proteinase inhibitor.

A major structural feature of the alpha-2-macroglobulin-like inhibitor of hagfish described in Osada, Nishigai, and Ikai [(1987) J. Ultrastruct. Mol. Struct. Res. 96, 00-00] was its highly open quaternary structure observed under an electron microscope. We drew a qualitative conclusion that the high frictional ratio obtained from the result of sedimentation study and the large Stokes radius obtained in gel chromatographic experiment were the reflection of such an open quaternary structure. In this paper I present several structural models of hagfish inhibitor based on its electron micrographs and calculate expected frictional ratios for such models according to the method developed by Bloomfield and his co-workers. Their method allows the calculation of frictional coefficient of a body of an arbitrary shape by approximating it with a collection of small spheres. To test the validity of such a method, macroscopic models were built from plastic spheres or cylindrical capsules and their translational frictional coefficients were measured by the free-falling method under experimental conditions where the Reynolds number was between 10(-3) and 10(-4).

Animals↗
PubMedDate not supplied

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope.

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.

Air↗