Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Copper and nickel binding to canine serum albumin. A circular dichroism study

The binding of copper and nickel to canine serum albumin has been studied using circular dichroism. In the 320-700 nm region, only a single positive extremum was observed at about 664-667.5 nm for copper bound to canine serum albumin. The intensity of this extremum was found to increase until a Cu2+/albumin molar ratio of 3 was reached. Further addition of Cu2+ led to a decrease in ellipticity. The absence of any extrema in the 560-570 and 480-510 nm regions showed that histidines were not involved in copper binding to canine albumin. In the case of nickel, initial binding was found to take place at the N-terminal tripeptide. At higher nickel to albumin molar ratios, circular dichroism spectra indicated the presence sulfur containing ligands but showed no evidence for the involvement of histidines. Canine serum albumin was found to bind six or more Cu2+ and Ni2+ ions with affinities that are lower than for human or bovine serum albumin.     

Albumin index in spot urine from outpatients with noninsulin-dependent diabetes mellitus

The albumin index (mg/g . creatinine) was determined in untimed spot urine collected in the early morning from 92 randomly selected outpatients with noninsulin-dependent diabetes mellitus (NIDDM). The patients were divided into three groups: 49 patients with normo-albuminuria (albumin index less than 9.1), 24 with micro-albuminuria (albumin index between 9.1 and 100), and 19 with overt-albuminuria (albumin index over than 100). With diabetic duration, the frequency of the patients with overt-albuminuria was increased, but that with normo-albuminuria was decreased. The patients treated with only a diet almost showed normo-albuminuria. In contrast, micro-and overt-albuminuria were found more frequently in the patients treated with oral hypoglycemic agents or insulin. Micro- and overt-albuminuria were found more frequently in the patients with poor glycemic control than in those with good glycemic control. The urinary albumin index was significantly high in the micro-albuminuric patients with poor glycemic control. Similarly, micro- and overt-albuminuria were found more frequently in the patients associated with diabetic retinopathy or neuropathy than in those without diabetic complications. In addition, overt-albuminuria was found more frequently in the patients with hypertension. The urinary albumin index was significantly high in the overt-albuminuric patients with hypertension. In conclusion, the determination of the albumin index in spot urine may be outpatients with NIDDM.     

Structural differences between albumin A and albumin C of the house mouse,Mus musculus

Two albumins, albumin A from C3H mice and albumin C isolated from descendents of the wild mice in which the variant was first uncovered, were found to differ in their electrophoretic properties. Albumin C was shown to bind two more H⁺ ions than albumin A at pH 5.4. Peptide mapping after trypsin digestion revealed that albumin C had three peptides (TP-C1, TP-C2, and TP-C3) which were missing in albumin A. The latter likewise had a peptide (TP-A1) which was not found in albumin C. An amino acid analysis of the variant peptides suggests that TP-A1 had been split into TP-C1 and TP-C2 on digestion with trypsin, because a glutamic acid in TP-A1 was replaced by a lysine. This change would also appropriately alter the electrophoretic properties of albumin C. No obvious counterpart was discovered for TP-C3 of albumin C in albumin A.This work was supported by a grant from the National Research Council of Canada.     

Quantitative study of aluminum binding to human serum albumin and transferrin by a chelex competitive binding assay

Binding of aluminum to human serum albumin and transferrin was investigated using a competitive binding assay incorporating a cation exchange resin, chelex. Both albumin and transferrin were found to produce linear Scatchard plots of aluminum binding data over the aluminum and protein concentration ranges found in humans. Binding constants measured for albumin and transferrin were 1.96 and 0.515 microM, respectively.     

Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid

We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin-silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin-silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin-deficient mice is suggested.     

Association equilibrium of d-methamphetamine and l-methamphetamine with serum albumin

The association equilibria for complex formation between serum albumin (bovine, rat) and the optical isomers of methamphetamine (MAMP) was determined using an ultrafiltration method. It was found that serum albumin/d-MAMP and serum albumin/l-MAMP complexes had distinctly different Scatchard plots with bovine and rat albumin. The binding parameters of each association equilibrium were estimated from the Scatchard plots by Rosenthal's graphic method. This distinguished two kinds of specific binding sites in terms of the association equilibrium between bovine serum albumin and d-MAMP, and one binding site for rat serum albumin and d-MAMP. One specific binding site was found between serum albumin and l-MAMP in both bovine and rat. Molar ellipticities, [θ], of peaks were decreased in the CD spectra of the complexes formed between bovine serum albumin and d-MAMP or l-MAMP when compared with the CD spectrum of bovine serum albumin alone. However, no difference in [θ] was found between the CD spectra of the enantiomers of MAMP in the measured wavelength range. The non-specific binding site was distinct from the specific binding site and resulting from altered tertiary structure of the albumin molecule. Chirality 10:742–746, 1998. © 1998 Wiley-Liss, Inc.     

Isolation of serum albumin-synthesizing polysomes from rat liver

The procedures for the purification of rat liver polysomes synthesizing serum albumin was developed, employing the quantitative precipitin method with rat serum albumin as a carrier and its antibody, and ribonuclease inhibitor from rat liver. The addition of ribonuclease inhibitor to polysomes during the incubation with antibody was found to prevent their degradation. Under these conditions, about 12 % of the membrane-bound polysomes of rat liver was found in the specific precipitate of serum albumin and its antibody, while a negligible amount of free polysomes was precipitated. It is concluded that polysomes synthesizing serum albumin are isolated by this method.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Identification of a 130-kDa albumin in tuatara (Sphenodon) and detection of a novel albumin polymorphism

Electrophoretic, immunochemical, and protein sequence analyses were performed on plasma albumin of the tuatara (Sphenodon), a rare reptile endemic to New Zealand. The analyses revealed that, unlike other terrestrial vertebrates, tuatara do not seem to possess a 60- to 75-kDa plasma albumin. The common form of plasma albumin in this genus has an apparent molecular mass of 130 kDa, making it by far the largest albumin reported for any terrestrial vertebrate. Starch gel electrophoresis of samples from tuatara on 24 of the 30 islands inhabited by this genus resolved two forms of the 130-kDa albumin (albumins A and C). A third albumin of approximately 170 kDa (albumin B), reflecting a novel alloalbuminemia, was found in tuatara in three geographically isolated populations. Albumin A appears to be restricted to populations at the southern extremity of the tuatara's distribution, while albumin C was found in all but four (southern) populations. Possible explanations for the origin and distribution of these albumins are discussed.     

Composition of culture medium is more important than co-culture with hepatic non-parenchymal cells in albumin production activity of primary rat hepatocytes,and the effect was enhanced by hepatocytes spheroid culture in collagen gel

Co-culture of primary rat hepatocytes with hepatic non-parenchymal cells or sinusoidal endothelial cells for albumin production activity as an index of liver-specific function was studied. The co-cultures were effective for the expression and maintenance of albumin production activity. However, the co-culture effect was not observed when we used a suitable culture medium, which had already been reported to be sufficient for albumin production activity. Albumin production of dispersed cells in collagen gel culture was higher than that of spheroid culture. In addition, albumin production of spheroids in collagen gel culture was higher than that of spheroid culture and dispersed cell collagen gel culture with a suitable culture medium. We found that culture medium composition was more important than co-culture for expression and maintenance of albumin production. Furthermore, we found that cell–cell interaction was effective for the expression of albumin production, but heterotypic cell–cell interaction was not necessary.     

Peculiarities in structure and properties of rabbit blood serum and liver albumins in ontogenesis

Isoelectrofocusing and infrared spectroscopy were used to study blood serum and liver albumins in rats aged 30, 45, 90 days, 6 months and 2 years. The analysis of infrared and isoelectric spectra shows that the blood serum albumin as compared to the liver albumin is more basic albumin with a less hard structure. With age theses albumins are found to become similar. An assumption is advanced that a modified albumin participates in the processes of this albumin decay and synthesis.     

Precursor–product relationship between intrahepatic albumin and plasma albumin

Rats were injected with [(3)H]leucine, and at various times thereafter labelled albumin was isolated by electrophoresis from their livers and blood plasma. The specific radioactivity of each protein was determined by spectrophotometry and liquid-scintillation spectrometry. Intrahepatic albumin was shown to be identical with plasma albumin by its electrophoretic mobility and antigenicity. It was found that intrahepatic albumin was the direct precursor of plasma albumin. Comparison of their specific radioactivities showed that intrahepatic albumin attained a higher specific radioactivity before plasma albumin. When plasma albumin reached its maximum specific radioactivity, that of intrahepatic albumin had decreased to a similar value. Thereafter, the specific radioactivity of intrahepatic albumin remained lower than that of plasma albumin.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Human alpha-fetoprotein and albumin: differences in free sulfhydryl groups

The number of free sulfhydryl (SH) groups of both human alpha-fetoprotein (AFP) and albumin, isolated simultaneously from human cord serum, was determined by reacting the proteins with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). A higher number of SH groups was consistently found with AFP than with albumin. When both proteins were pretreated with 100 mmol/l 2-mercaptoethanol, only approximately 0.5 SH groups were detected per molecule of human adult albumin, yet as many as 4 per molecule were found with AFP. Dithiothreitol was found to be more effective than 2-mercaptoethanol in SH group regeneration. The results suggested that some disulfide linkages of AFP could be reduced. Free SH groups regenerated by the treatment with SH reagent were found to disappear gradually during storage under aerobic conditions. The rate of disappearance differed between AFP and albumin.     

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : II. THE RELATION BETWEEN NUMBER OFSH AND S-S GROUPS AND QUANTITY OF INSOLUBLE PROTEIN IN DENATURATION AND IN REVERSAL OF DENATURATION

1. In native egg albumin no SH groups are detectable, whereas in completely coagulated albumin as many groups are detectable as are found in the hydrolyzed protein. In egg albumin partially coagulated by heat the soluble fraction contains no detectable groups, and the insoluble fraction contains the number found after hydrolysis. 2. In the reversal of denaturation of serum albumin, when insoluble protein regains its solubility, S-S groups which have been detectable in the denatured protein, disappear. 3. When egg albumin coagulates at an air-water interface, all the SH groups in the molecule become detectable. 4. In egg albumin coagulated by irradiation with ultraviolet light, the same number of SH groups are detectable as in albumin coagulated by a typical denaturing agent. 5. When serum albumin is denatured by urea, there is no evidence that S-S groups appear before the protein loses its solubility. 6. Protein denaturation is a definite chemical reaction: different quantitative methods agree in estimates of the extent of denaturation, and the same changes are observed in the protein when it is denatured by different agents. A protein molecule is either native or denatured. The denaturation of some proteins can be reversed.     

Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.     

Tryptophan content of serum albumin

Tryptophan content of serum albumin was determined spectrophotometrically. The method used for the determination of tryptophan gave consistent results. Results show that the tryptophan contents of bovine and human serum albumin are significantly different from chicken serum albumin. Bovine and human serum albumins, however, are not significantly different from each other. A large difference in tryptophan content was found between two samples of chicken serum albumin. This suggests that the tryptophan content of serum albumin may not be constant for any given species. For these reasons, tryptophan content should not be used to estimate the molecular weight of serum albumin.     

Albumin Paris 2: a new genetic variant distinguished by isoelectric focusing.

Until recently, the characterization of genetic variants of human serum albumin was performed by electrophoretic typing prior to the determination of their amino acid substitutions. We describe a procedure using isoelectric focusing in the presence of urea for the analysis of the genetic variation of albumin. This procedure allowed a clear distinction of a new variant, previously found to be identical with albumin Sondrio according to its relative electrophoretic mobilities at 3 pHs. This new variant, the third rare albumin allotype identified in the Ile-de-France region, was called albumin Paris 2.     

Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and secretion of serum albumin

A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones.The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.