S-Transnitrosylation of albumin in human plasma and blood in vitro and in vivo in the rat

S-Nitrosoalbumin (SNOALB) is the most abundant physiological circulating nitric oxide (NO) carrier regulating NO-dependent biological actions in humans. The mechanisms of its formation and biological actions are still incompletely understood. Nitrosation by authentic NO and S-transnitrosylation of the single sulfhydryl group located at Cys-34 of human albumin by the physiological S-nitroso compounds S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO) are two possible mechanisms. On a quantitative basis, we investigated by gas chromatography-mass spectrometry the contribution of these two mechanisms to SNOALB formation in human plasma and blood in vitro. GSNO and SNOC (0-100 microM) rapidly and efficiently (recovery=35%) S-transnitrosylated albumin to form SNOALB. NO (100 microM) S-nitrosated albumin to SNOALB at a considerably lower extent (recovery=5%). The putative NO-donating drugs glyceryl trinitrate and sodium nitroprusside (each 100 microM) failed completely in S-nitrosating albumin. Bubbling NO into human plasma and blood resulted in formation of SNOALB that inhibited ADP-induced platelet aggregation. Infusion of GS(15)NO in the rat resulted in formation of S(15)NOALB, [(15)N]nitrate and [(15)N]nitrite. Our results suggest that S-transnitrosylation of albumin by SNOC and GSNO could be a more favored mechanism for the formation of SNOALB in the circulation in vivo than S-nitrosation of albumin by NO itself.     

Binding of aflatoxin B1, G1 and M to plasma albumin

The aflatoxins B₁, G₁ and their metabolites exist in the systemic blood as protein conjugate. This conjugation is specific to plasma albumin and proceeds enzymatically by liver and kidney cells. The aflatoxin-albumin conjugate is permanent and the conjugation is an irreversible one. This may interpret the acute liver damage of animal ingested a single dose of aflatoxin (3, 4). The existence of bound aflatoxin-albumin in the systematic blood could be considered as one factor of low excretions of aflatoxins and their metabolites in urine (5, 6, 7).     

Binding of copper to albumin and participation of cysteine in vivo and in vitro

Albumin, the major copper-binding protein in blood serum, was shown to form different albumin-copper complexes in in vivo and in vitro. Cupric ions added in vitro to control rat serum bound preferentially to mercaptalbumin and the mercaptalbumin-copper complex remained unchanged with time. Cupric ions injected intravenously into the rat first formed the mercaptalbumin-copper complex; this binary complex changed gradually with time to form an albumin-copper-cysteine complex. The participation of cysteine in the formation of this complex was demonstrated in vitro and further suggested that its conversion was an oxidative reaction. Glutathione also participated in forming the complex, but it was not as effective as cysteine. Albumin-copper complexes were separated on a gel filtration column and detected simultaneously by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry.     

Cytological studies of plasma albumin in the rat liver

Summary The presence of albumin in the parenchymal cells of the rat liver is demonstrated using the fluorescent antibody method. It is evident that there is no immunological difference between liver and blood albumin. The liver section is not uniformly stained by the fluorescent antibody and this may be due to the uneven distribution of albumin in the liver. A small percentage of the parenchymal cells presumably containing albumin are markedly fluorescent. The nuclei of these cells are not fluorescent implying the absence of albumin and this is consistent with the fact that nuclei are not involved in synthesis of albumin. The scattered distribution of brightly fluorescent cells confirms earlier observations that all parenchymal cells are not functionally alike, although they appear homogenous histologically.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

Influences of heparin on ACTH distribution and immunoreactivity in plasma of the rat. In vivo and in vitro studies

1. Blood samples from non-pregnant female rats were incubated in vitro with porcine 125I-ACTH, and the corresponding plasmas were chromatographed on fine Sephadex G 50. When heparin was added in vivo or in vitro, almost all the radioactivity appeared in the void volume of the columns; the same was observed when labelled ACTH was added to heparin-containing saline. In contrast, when NaCl instead of heparin was added to the blood in vivo as well as in vitro, almost all the plasma radioactivity was eluted later, with 125I-ACTH. 2. When labelled ACTH was i.v. administered to pregnant females, it was eluted in the void volume in the presence of heparin, and further down in its absence. 3. The same plasma samples from non-stressed or ether-stressed females were radioimmunoassayed for ACTH, with and without heparin. The degradation of ACTH was greater in the presence of heparin, and plasma ACTH concentration was understimated for low blood levels of heparin (5 UI/ml or less) and in contrast overestimated for high ones (25 or 50 UI/ml). 4. In conclusion, the reported data clearly demonstrated firstly that heparin added to rat blood traps ACTH molecules, promoting the formation of aggregates with apparent height molecular weight; secondly that heparin interferes with the direct radioimmunoassay of ACTH in the plasma.     

Zinc transport by transferrin in rat portal blood plasma.

Zinc transport in mesenteric lymph and zinc distribution in portal plasma and venous plasma were examined in rats that had been given an oral dose of ⁶⁵Zn. Less than 1% of an oral dose of ⁶⁵Zn appeared in the mesenteric lymph over a period of 8 hr. In portal plasma, approximately 70% of the isotope recovered after gel-filtration chromatography was bound to a protein that was identified as transferrin on the basis of molecular weight and electrophoretic properties. In venous plasma, the major fraction of ⁶⁵Zn was bound to albumin while the remainder of the isotope was associated with higher molecular weight proteins including transferrin and α₂-macroglobulins. These results demonstrate that zinc is transported from the intestine to the liver via the portal blood, and the results demonstrate that zinc is transported in portal plasma bound to transferrin.     

Spectroscopic studies of the interaction between hypocrellin B and human serum albumin

Previous work has proved that hypocrellin B (HB) binds to human serum albumin (HSA) at a specific site instead of distributed randomly on the surface of a protein. In the current work, further investigation by using bilirubin as a site I marker indicates that HB can compete for the same site with bilirubin, suggesting that the HB binding site is located at sub-domain IIA (site I) of HSA. Moreover, bound to HSA, the HB fluorescence was found to be pH sensitive in physiological range (pH 6.0-8.0). The increasing of binding constant of HB to HSA in the pH range 6-8 also indicates that the N<-->B transition modulates the microenvironment changes of the binding site and influences considerably the binding between HB and HSA. Furthermore, picosecond time-resolved fluorescence spectra of HB-HSA complex in PBS indicate an additional short-lived component compared to that for HB in benzene, which may be assigned to the process of electron transfer from Trp-214 to HB.     

Binding of epididymal proteins to rat spermatozoa in vivo.

The secretion of epididymal proteins and their binding to spermatozoa in rats were examined after retrograde perfusion of the superior and inferior epididymal arteries with [35S]methionine. PAGE revealed that the pattern of radioactive proteins in the luminal fluid was markedly different from the well-characterized pattern of secretory proteins obtained by in vitro incubation of epididymal minces with labeled methionine. Of the proteins secreted into the lumen, about 1% were associated with Percoll-purified spermatozoa. More proteins were associated with the spermatozoa in the corpus epididymidis than in the caput. Sequential extraction of spermatozoa with an isotonic buffer, a high-salt buffer, Triton X-100, and SDS revealed that almost half of the radiolabeled proteins could be extracted with the isotonic buffer. The firmly bound radioactive proteins remaining, which were extracted with Triton X-100 or SDS, consisted of one major band of 25 kDa and two minor bands of 30 kDa and 32 kDa. Analysis of the sperm-associated proteins at various times after the isotope was administered indicated that tight binding of proteins to spermatozoa occurs within 3 h after isotope injection.     

Binding of estrogen-3-sulfates to stallion plasma and equine serum albumin.

The binding of estrone-3-sulfate (E1-3-S) and estradiol-3-sulfate (E2-3-S) to adult stallion plasma was determined and compared with the binding to equine serum albumin (ESA). On the ESA molecule, two binding sites for E1-3-S with an association constant of 1.3 x 10(5) M-1 and several sites of weaker affinity were found; the data for E2-3-S showed the existence of four binding sites of moderate affinity (1 x 10(5) M-1) and several sites of weaker affinity. The removal of albumin from the stallion plasma resulted in the absence of binding of E1-3-S or E2-3-S, whereas the removal of glycoproteins resulted in binding parameters similar to those obtained with whole plasma. These results indicate that ESA is the only estrogen sulfate binder in horse plasma. Under physiological conditions, 95% of E1-3-S was bound to ESA.     

Binding of phytochrome to plasma membranes in vivo

A microsomal fraction was prepared by differential centrifugation from the homogenate of dark grown shoots of oats ( Avena sativa L. cv. Sol II). Plasma membranes were prepared from the microsomal fraction by means of an aqueous polymer two phase partition method. The content of phytochrome in the microsomal fraction and the plasma membrane fraction, respectively, were studied after different irradiation treatments of the intact shoots. Red irradiation increased the content of phytochrome in both the microsomal and plasma membrane fraction, especially in the presence of Mg²⁺. The increase induced by red light was fully reversible by far-red light for the plasma membrane fraction both in the presence and absence of Mg²⁺, in contrast to the microsomal fraction where Mg²⁺ had to be omitted. KI treatment of the membranes destroyed the binding of phytochrome whereas agents such as KCI, EDTA, CaCl₂ and Triton X-100, did not have this effect, indicating that the phytochrome attachment to the membrane is hydrophobic. This in vivo binding resembles to a large extent the one obtained in vitro by Sundqvist and Widell (Physiol. Plant. 59: 35–41, 1983) even though some differences between the phytochrome species and the membrane side exposed probably occur; so that the present interaction between phytochrome and the plasma membrane does not necessarily reflect the interaction that leads to physiological responses, and there could be more than one type of interaction.     

In vivo and in vitro studies of hafnium-binding to rat serum transferrin

The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.     

Fluorescence studies of the interactions of serum albumin and rat alpha1-fetoprotein with aflatoxin B1. Specificity and binding parameters.

The interaction of bovine serum albumin and rat alpha1-fetoprotein with aflatoxin B1 has been followed by the fluorescence quenching of the protein in presence of the ligand. The binding parameters (n, number of sites and Kd, dissociation constant) have been determined for the bovine serum albumin-alflatoxin B1 system: n = 3.5 and Kd = 3.1 +/- 0.5 . 10(-5) M and for the alpha-fetoprotein-aflatoxin system: n = 4 and Kd = 3.7 +/-0.5 . 10(-5) M. The competition of anilino-naphthalene-sulfonate and aflatoxin B1 for the same hydrophobic sites on bovine serum albumin has been demonstrated. The fluorescence quenching of various proteins (lysozymes, egg-albumin, gamma-globulin) by aflatoxin B1 have shown that there is not a strict specificity of aflatoxin towards proteins.