The control of serum protein synthesis in hepatoma-fibroblast hybrids.

Hybrids between mouse hepatoma cells (which secrete several serum proteins) and mouse or rat fibroblasts (which do not secrete these proteins) produce transferrin and the third component of complement (C3) like the parental hepatoma cells, while they do not secrete either albumin or alpha-fetoprotein (AFP). This lack of albumin and AFP secretion is probably due to a lack of synthesis, rather than to a simple defect in secretion. The cessation of albumin and AFP production is not dependent upon the parental fibroblast nor upon the selection conditions; it is best explained by a shut-off synthesis and could thus reflect the existence of a regulatory mechanism. This would imply a difference between the control of albumin and AFP synthesis and that of transferrin and C3 synthesis. On the other hand, in agreement with Peterson and Weiss (1972), hybrids between rat hepatoma cells and mouse fibroblasts continue to product rat albumin. This suggests that the mouse hepatoma cells differ from the rat hepatoma cells in the way they control albumin production.     

The 95000-Mr gelatin-binding protein in human serum and plasma.

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.     

The regulation of protein synthesis in animal cells by serum factors.

We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.     

The location of protein in serum lipoproteins: a fluorescence quenching study.

Quenching of the tryptophan fluorescence of pig serum HDL3 and LDL2 lipoproteins by iodide and succinimide has been used to estimate the accessibility of the fluorophores to the solvent and, by inference, the location of the protein in the macromolecular complexes. At least 80% of the protein is thought to be located at or near the surface in both lipoproteins but its accessibility is hindered especially in LDL2. A difference in surface topography in the two lipoproteins is suggested with the protein in LDL2 more buried in lipid and further away from the charged phospholipid polar groups than in HDL3. A refined treatment of the quenching data has been developed to take account of the heterogeneity of quenching sites found in the lipoproteins.     

Multivalent ligand binding by serum mannose-binding protein.

The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP.     

Role of human serum biotinidase as biotin-binding protein.

Biotinidase shows two binding sites for biotin, with Kd = 59 and 3 nM respectively, and requires tryptophan and cysteine residues of the biotinidase protein for biotin-binding activity. Analysis of human serum by various column-chromatographic techniques indicates that biotinidase is the only protein which exchanges with labelled (+)-biotin. It was shown previously that epileptic patients receiving a high average dose of anticonvulsants (containing a carbamide group) have lower biotin concentrations than those receiving a low dose. We have shown in human serum and with purified biotinidase that these anticonvulsant drugs compete with biotin for binding to the protein moiety.     

Effect of rhythmic alternation of protein depletion and repletion on serum protein synthesis.

The authors attempted to determine whether repeated protein depletion would produce changes in the organism's response to this unfavourable situation indicative of the preservation of information on past depletion and of its inclusion in the formation of the organism's defences against repetition of this unphysiological state. It can be concluded from the results that the anticipated trend was demonstrated. The question of the site of origin, storage and action of this information was not resolved.     

The distribution of J blood-group activity on lipoprotein and protein fractions of bovine serum.

There is a diversity of carriers of the J blood-group activity of bovine serum. The qualitative and quantitative distribution of the J activity on different carriers was studied, using various fractionation procedures. Approximately one third of J activity was found in the total lipids extracted from serum, two thirds in the lipid-free residue precipitated by lipid extraction. One third of the lipid J substance was found to be bound to the very low density lipoprotein, two thirds to the low density lipoprotein, while the high density lipoprotein was completely free of J activity. All non-lipidic J substance was present in the lipid-free protein. There was no J activity in the low molecular weight mucoproteins of serum and in the apoproteins of the lipoprotein fractions. The lipoprotein fractions were prepared by ultracentrifugation at different solvent densities. The lipoprotein fractions were characterized by chemical analyses and physical properties. The lower total cholesterol concentration of bovine serum, as compared to human serum, is reflected in a lower concentration of low density lipoprotein. The results obtained by ultracentrifugation coincide with the results obtained by precipitation of "beta-lipoproteins" with dextran sulfate and calcium chloride and with results obtained by gel filtration of bovine serum. The "beta-lipoprotein" fraction contains lipoproteins of very low and low density, and probably chylomicrons and a variety of other proteins, however no high density lipoprotein.     

Isolation of mouse C-reactive protein from liver and serum.

Purified proteins have been isolated from the sera and livers of mice. These proteins are antigenically identical but share antigenic determinants in common with human C-reactive protein and do not share antigenic determinants in common with mouse or human immunoglobulins. The proteins interact with C-polysaccharide, are precipitated by calcium ions, migrate electrophoretically with gamma mobilities, and have isoelectric points of 4.8 and 5.62. Since these properties are characteristic of C-reactive protein of man, monkey, rabbit and dog, the pure proteins isolated from mice are designated mouse C-reactive protein.     

Ultracentrifugation evidence for a somatostatin-binding protein in serum.

Using a technique of high speed centrifugation of serum and a well validated immunoassay for the measurement of serum somatostatin-like immunoreactivity, we have demonstrated that somatostatin, unlike other peptide hormones, appears to sediment with large molecular weight proteins. When synthetic somatostatin of increasing concentration was incubated with serum prior to ultracentrifugation, a linear plot of concentration of somatostatin added against concentration sedimenting (or apparently bound to protein) revealed an association curve. These data provide further evidence for the existence of a serum-binding protein for somatostatin.     

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum.

The presence of O-acetyl-5-methoxytryptophol in the pineal glands of rats kept in the dark for 8 h, but not in the light, has been shown by means of g.l.c.-mass spectrometry. It is suggested that this compound may be the biologically active precursor of circulating methoxytryptophol.