Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation.

The conformation of the fully disulfide-reduced state of human serum albumin was investigated by tryptophan fluorescence spectrum, CD analyses, and size-exclusion chromatography. Both the reduction of the native disulfide-bonded form under nondenaturing conditions and the refolding of the urea-denatured disulfide-reduced form under reduced conditions yielded almost exactly the same disulfide-reduced state with partially folded unique conformation that was clearly distinguished from either the native or fully denatured state. In addition, the interconversion between the urea-denatured reduced form and the partially folded reduced form was reversible with each other; by reoxidation, the partially folded reduced form was converted to the disulfide-bonded form. The conformation of disulfide-reduced serum albumin was highly variable depending on pH and ionic strength conditions. Thus, we concluded that the disulfide-reduced state with partially folded variable conformation is involved in the reversible interconversion between the denatured reduced form and the native disulfide-bonded form of human serum albumin.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Local formation and degradation of endothelin-1 in guinea pig airway tissues

Endothelin(ET)-1 and big ET-1 caused potent and sustained constriction of isolated guinea pig bronchus. The response to ET-1 was enhanced by phosphoramidon in a simple dose-related manner (0.01-1000 microM), while the response to big ET-1 was enhanced at lower doses (0.01-0.1 microM) but was suppressed at higher doses (100-1000 microM) of phosphoramidon. Big ET-1, when given intravenously (i.v.) to anesthetized guinea pigs, increased both bronchopulmonary inflation pressure and mean arterial blood pressure (2.5, 5, 10 nmol/kg i.v.). The pressor response to big ET-1 was attenuated by phosphoramidon dose-relatedly, while the pulmonary response was modified in a complex fashion composed of delayed onset and prolonged duration of action. These results suggest that ET converting as well as degrading enzymes coexist in the airway tissue and both enzymes are sensitive to phosphoramidon, so that phosphoramidon acts bifunctionally to reduce and stimulate the airway responses to big ET-1.     

Crystallization of and preliminary crystallographic data for the N-terminal half-molecule of ovotransferrin

The N-terminal half molecule of ovotransferrin has been crystallized from a polyethylene glycol 6000 solution by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with cell dimension of a = 47.0, b = 90.2, and c = 76.2 A. The crystals diffract X-rays to a resolution limit of at least 2.0 A and are resistant to X-ray radiation damage. They appear to be suitable for X-ray structure analysis.     

Phalloidin-induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.     

Polyamine prevention of inhibition of rat liver isoleucyl-tRNA formation by poly(G), poly(I) or ribosomes.

It is shown that rat liver isoleucyl-tRNA formation in the presence of Mg²⁺ is inhibited by poly(G), poly(I) or ribosomes and that this inhibition is prevented by polyamines. The inhibition is found to be noncompetitive with respect to tRNA.     

Change of substrate specificity by polyamines of ribonucleases which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides.

The activities of ribonucleases (RNase HS and RNase A), which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides were stimulated by polyamines, while the activities of ribonucleases (RNase T₁ and RNase M), which attack ribonucleic acid at linkages attached to purine nucleotides were not influenced by polyamines. In the presence of polyamines, the cleavage of C_5′-O-P linkages adjacent to cytosine nucleotide was stimulated, while the cleavage of C_5′-O-P linkages adjacent to uracil nucleotides was inhibited slightly. The effect of polyamines on the activities of ribonucleases occured through the binding of the polyamines to nucleic acid.