Polymorphism in fowl serum albumin. VII. Distribution and activity of free and membrane-bound polysomes in developing fowl liver

The quantity and activities of membrane-bound and free polysomes in livers from chick embryos at successive stages of development were compared in cell-free protein-synthesizing systems. Membrane-bound polysomes increased 2-fold between 8 and 18 days of development, while total ribosome content remained constant. Free polysome activity also remained constant during this period, while that of membrane-bound (total--free) polysomes decreased, possibly because of an increase in ribonuclease activity in this fraction. Serum albumin biosynthesis occurred primarily on membrane-bound polysomes. With liver development, increased secretion of serum proteins may be correlated with synthesis of serum albumin on increasing numbers of membrane bound polyribosomes.     

Polymorphism in fowl serum albumin. VI. Changes in in vitro protein synthesizing activity in developing embryonic fowl liver

Summary Cell-free protein synthesizing systems were prepared from the livers of chick embryos at selected ages and the characteristics of individual fractions were compared. While polysomes showed decreasing size with older embryos, isolated polysomes did not differ significantly in amino acid incorporating activity when assayed with standard cell sap. When assayed with standard polysomes, cell sap activity decreased with increasing developmental age whether incorporation was measured using [³H]lysine, [³H]leucine, or [³H]aminoacyl-tRNA. Free amino acid concentrations in the cell sap showed reproducible independent variation during development which was taken into consideration in calculating net amino acid incorporation. A large increase in ribonuclease activity was observed during development; however, nuclease inhibitor activity was absent before day 15 but increased thereafter. Aminoacyl-tRNA synthetase activity did not vary significantly. It is proposed that the observed changes in the rate of cell-free protein synthesis result not only from increasing ribonuclease activity with increasing developmental age but also from changes in the activity of other soluble factors.This is paper VI in a series; paper V is reference 6. The series title is based on earlier work with systems derived from fowl which synthesized two genetic variants of serum albumin²¹.This research was supported in part by a grant from the Damon Runyon Memorial Fund (DRG-1125). Dr. H. M. Jernigan was an N.I.H. Postdoctoral Fellow (5 F02 GM 50944-02).To whom all inquiries are to be addressed.     

Raman studies of bovine serum albumin.

The Raman Spectra of bovine serum albumin have been obtained in the solute state, in alkaline and acidic solutions, and in the gel. The reversible denaturations of bovine serum albumin solutions by heat, acid's, and alkali were studied and a new mechanism for heat denaturation has been proposed based on a continuous unfolding of the α-helices.     

Large fragments of human serum albumin.

Large fragments of human serum albumin were produced by treatment of the native protein with pepsin at pH3.5. Published sequences of human albumin [Behrens, Spiekerman & Brown (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591; Meloun, Moravek & Kostka (1975) FEBSLett.58, 134-137]were used to locate the fragments in the primary structure. The fragments support both the sequence and proposed disulphide-linkage pattern (Behrens et al., 1975). As the pH of a solution of albumin is lowered from pH4 to pH3.5, the protein undergoes a reversible conformational change known as the N-F transition. The distribution of large fragments of human albumin digested with pepsin in the above pH region was critically dependent on pH. It appeared that this distribution was dependent on the conformation of the protein at low pH, rather than the activity of pepsin. The yields of the large fragments produced by peptic digestion at different values of pH suggested that the C-terminal region of albumin unfolds or separates from the rest of the molecule during the N-F transition. The similarity of peptic fragments of human and bovine albumin produced under identical conditions supports the proposed similar tertiary structure of these molecules.     

Genetic variants of serum albumin: a study of albumin Kashmir.

The study of human serum albumin variants is reviewed with reference to albumin Kashmir, a typical variant. Its published instances are listed and its position in this field of investigations is indicated.     

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.     

Heat-ethanol isolated serum albumin: discussion.

Albumin isolated by the Hagen/Münster heat-ethanol method is a remarkably stable solution of high purity and clinical compatibility. The simplicity of this method makes it a model fractionation procedure for the small transfusion service. Immunization studies carried out by us are compared with previous accounts of a "new" antigen occuring in plasma protein solution produced by partial heat denaturation. Clinical compatibility of cold-ethanol and heat-ethanol fractionated albumin solutions are also shortly discussed.     

Summary of recombinant human serum albumin development.

The methylotrophic yeast Pichia pastoris is well known as a host strain for the production of a variety of heterologous proteins. We have used P. pastoris for the production of recombinant human serum albumin (rHSA), for which we have developed efficient and specialized downstream processes. Results from structural analysis suggest that purified rHSA possesses an identical conformation to plasma derived human albumin (pdHA) and no difference from pdHA has been observed in neo-antigenicity. Host-cell-derived impurities (i.e. Pichia yeast component, DNA and mannan) have been evaluated in the purification process as well as in the drug substance and relevant specifications established. The efficacy and safety of rHSA have been tested in clinical studies and no difference from pdHA has been found in comparative study. Such studies have confirmed rHSA to have high efficacy with little or no adverse reaction.     

Freeze-drying of solutions of serum albumin containing dimethylsulfoxide.

Although several publications have emphasized the inadvisability of drying biologic materials containing dimethylsulfoxide (DMSO) by sublimation of ice in vacuo, our studies showed a relationship to exist between the relative concentrations of serum albumin, human, and dimethylsulfoxide for successful or unsuccessful freeze-drying of albumin-DMSO solutions. The cycle of freeze-drying for the successful drying of an albumin-DMSO solution was a modification of the cycle used for the successful drying of suspensions of measles virus by sublimation of ice in vacuo. Using nuclear magnetic resonance spectroscopy, a strong, sharp signal for DMSO was obtained in preparations of freeze-dried albumin-DMSO solutions rehydrated with deuterium oxide, D₂O.     

Bovine serum albumin in lithium chloride solutions.

The effects of li+ and H3O+ on the conformation of bovine serum albumin in azqueous solutions at room temperature are compared. At low pH (high concentration of H3O+) the change in conformation of the protein is demonstrated by an increase in effective volume, a decrease in helical content and a blue shift of tyrosyl residue. A similar change is observed for the protein in highly concentrated LiC1 solution (6.0-7.0M) at neutral pH. However, the H3O+ is 12,000 times more powerful than the Li+ in destabilizing the protein molecule. This is consistent with their thermodynamic and kinetic properties, since the H3O+ is often different from the Li+ in several orders of magnitude. While the changes in structural properties of the protein are almost identical in both the acidic solution and the highly concentrated LiC1 solution, further study using dioxane as a probe suggests different mechanisms under which the changes occur. The effect of H3O+ is related to electrostatic force, whereas the effect of Li+ is related to both the electrostatic hydrophobic forces. These two major forces are believed to be responsible for the conformation of protein molecules.     

Immunochemical studies of fragments of bovine serum albumin.

Antigenic properties of 14 fragments of bovine albumin were measured using antisera to albumin and to two of its fragments. All seven fragments larger than 21,000 daltons formed immune precipitates. Although immune precipitates were not formed with smaller fragments, inhibition tests indicated the presence of antigenic sites on several of these fragments as well. The results predict the occurrence of six or more antigenic determinants and allow assignment of their positions in the parent molecule. These sites are distributed along the entire protein chain, with the sites of greatest antibody affinity situated in the COOH-terminal region. Evidence is presented that some sites are homologous, reacting with the same populations of antibodies, and that other sites are unique, binding to an exclusive population of antibodies.