The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.     

The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.     

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.     

Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine in-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1.5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal alpha-amino group, as well as lysine in-amino groups, are combined with chlorogenoquinone.     

Effects of microbial proteinase inhibitors on the degradation of endogenous and internalized proteins by rat yolk sacs.

1. The effects of leupeptin and other microbial proteinase inhibitors were measured in rat yolk sacs on the uptake and degradation of formaldehyde-denatured 125I-labelled bovine serum albumin as well as on the degradation of 3H-labelled endogenous protein. 2. Leupeptin, at concentrations between 1 and 100 micrograms/ml, inhibits the degradation of added albumin without affecting pinocytic uptake. Accordingly large amounts of undegraded albumin accumulate within the tissue. 3. Removal of leupeptin produces a rapid recovery of the capacity to degrade albumin. 4. Endogenous protein degradation is rapidly inhibited by leupeptin, but to a far lesser extent than the breakdown of albumin. However, the inhibition is only slightly reversed on removal of leupeptin. 5. Degradation of both albumin and endogenous protein in intact yolk sacs is inhibited by the microbial proteinase inhibitors in the order: leupeptin greater than antipain greater than chymostatin; elastatinal, pepstatin and bestatin are ineffective. 6. Similar results are found when albumin is incubated in yolk-sac homogenates at pH 4 with the inhibitors. 7. The marked inhibitory effects of leupeptin, antipain and chymostatin suggest that cathepsin B and possibly cathepsin L participate in the degradation of 125I-labelled albumin in yolk sacs. By comparison, the smaller inhibitory effects of the proteinase inhibitors on endogenous protein breakdown imply a minor role of lysosomal cathepsins in this process.     

Changes in the antioxidant properties of substituted phenol polydisulfides during interaction with human serum albumin

Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) in aqueous solutions were shown to form polycomplexes with human serum albumin. This process was accompanied by considerable changes in the spectrum of protein circular dichroism recorded in distilled water in the far UV range at 20 degrees C. Complex formation between human serum albumin and polydisulfides was followed by a marked decrease in the content of alpha-helices and increase in the count of antiparallel beta-structures in the protein. Stable complexes containing 1.5, 2.8, and 7.7 poly(2-aminodisulfide-4-nitrophenol) molecules per human serum albumin molecule were formed in bicarbonate buffer (pH 9.0). In these complexes, the secondary protein structure underwent changes similar to those in polycomplexes of human serum albumin and polydisulfides. Gallic acid polydisulfide and poly(2-aminodisulfide-4-nitrophenol) inhibited the catalase-induced degradation of 50 mM H2O2. Complexes of human serum albumin and poly(2-aminodisulfide-4-nitrophenol) increased the catalytic activity and operational stability of catalase 1.5 and 4-7-fold, respectively. This was characterized by the effective reaction rate constant (kin, s-1). Our results indicate that complexes of human serum albumin and substituted phenol polydisulfides act as potent protectors and activators of catalase during enzymatic degradation of H2O2 at high concentrations.     

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.     

Regulation of protein degradation in normal and transformed human cells. Effects of growth state, medium composition, and viral transformation.

In WI-38, a normal human fibroblast, the rates of degradation of short lived and long lived proteins are identical whether the cultures are growing exponentially or are density-inhibited. Replacement of the growth medium with fresh medium does not alter these rates. In VA-13, an SV-40 transformed derivative of WI-38, the rates of protein degradation are also independent of growth rate and fresh medium. However, in both WI-38 and VA-13 the rate of long lived protein degradation increases as the serum concentration is reduced below 5%. After complete serum withdrawal, the rate increases by 60 to 100% in both cell types. Withdrawal of arginine and phenylalanine triples the rate of long lived protein degradation, while addition of 10% dialyzed serum to this amino acid-deficient medium reduces the effect to twice that of the controls. Incubation of both types of cells in phosphate-buffered saline also increases protein degradation. This effect is reduced by glucose, albumin, and dialyzed serum. Therefore, the rate of protein degradation is independent of growth rate in normal and transformed human cells. However, the rate of degradation is closely coupled to certain medium alterations.     

Phosphatidyl choline interaction with bovine serum albumin: effect of the physical state of the lipid on protein-lipid complex formation.

Radioactively labeled [¹⁴C]phosphatidyl choline dispersed on Celite was equilibrated with bovine serum albumin solutions buffered at pH 8.0. Phosphatidyl choline was rapidly solubilized in the presence of serum albumin, and formed stable protein-lipid complexes which were isolated by gel-filtration through a Sepharose 4B column. Under similar conditions, equilibration of the protein with phosphatidyl choline liposome dispersions in buffer did not result in complex formation. The altered physical state of phosphatidyl choline on the weakly adsorbing Celite surface appears to be essential for binding by native bovine serum albumin. This work reports the first observation of phosphatidyl choline binding to native serum albumin in bulk phase and suggests the possibility of exposing monodisperse lipids, under controlled conditions, to proteins having lipid binding properties.     

Changes in erythrocyte and thrombocyte aggregation after ultraviolet irradiation

Lipid peroxidation which occurs in blood serum under ultraviolet irradiation was studied. The products of these reaction suppress ADP-induced aggregation of native platelets. The rouleaux-forming capacity increased after UV-irradiation of plasma and serum albumin. Under UV-irradiation the aggregates of albumin molecules are supposed to form the aggregates of albumin molecules which bind the erythrocytes in rouleaux.     

Polypeptide linkages and resulting structural features as powerful chromogenic factors in the Lowry phenol reaction. Studies on a glycoprotein containing no Lowry phenol-reactive amino acids and on its desialylated and deglycosylated products.

With bovine serum albumin as the reference standard, the armadillo salivary-gland glycoprotein, although containing no chromogenic amino acids and only small amounts of colour-yielding peptides [Chou & Goldstein (1960) Biochem. J. 75, 100-115], is highly reactive in the Lowry phenol protein assay [Wu & Pigman (1977) Biochem. J. 161, 37-47]. After desialylation and Smith degradation of the glycoprotein, the Lowry phenol value increased by 13 and 30% respectively, which suggests that both sialic acid and N-acetylhexosamine exert shielding effects in this reaction. Acid hydrolysis for 30 min decreased the Lowry phenol value by more than 45%, which indicates that the peptide linkages and steric features affect the Lowry phenol reactivity. After hydrolysis for up to 6h, the remaining Lowry phenol value of the partially hydrolysed core protein paralleled the amount of unhydrolysed peptides, inferring that both acid-sensitive and acid-resistant chromophoric peptides are fairly evenly distributed along the whole polypeptide chain. As with bovine serum albumin, more than 80% of the colour yield obtained in the Lowry phenol assay with this glycoprotein is Cu2+-dependent.     

Role of thiols, pH and cathepsin D in the lysosomal catabolism of serum albumin.

Attempts were made to assess the role of thiols and to determine the cathepsins involved in the degradation of serum albumin in mouse liver and kidney lysosomes. Unlike cysteine or beta-mercaptoethanol, reduced glutathione (GSH) did not stimulate the degradation of formaldehyde-treated albumin in liver lysosomes, suggesting that the tripeptide did not penetrate the membrane. However, GSH was a much more effective stimulant of proteolysis in kidney lysosomes than was cysteine at low concentrations, and the effect was saturable at 1-2 mM concentrations. Thiols did not stimulate proteolysis in lysosomes when the disulphide bonds of albumin were reduced and alkylated, suggesting that the stimulatory effects were solely due to disulphide-bond reduction in protein substrates. Results obtained with thiols and iodoacetamide suggested that albumins denatured by disulphide-bond reduction and alkylation, disulphide-bond reduction without alkylation, or by treatment with 8 M-urea, were all degraded primarily by cathepsin D in lysosomes, but formaldehyde-denatured albumin was attacked by thiol proteinases. These findings correlated well with studies on the degradation of these proteins by rat liver lysosome (tritosome) extracts. Studies with the proteinase inhibitors leupeptin and pepstatin and the stimulatory effects of thiols in these extracts suggested that formaldehyde-denatured albumin was degraded primarily by the thiol proteinases, but that native albumin or albumins denatured by disulphide-bond reduction or by treatment with 8 M-urea were attacked by cathepsin D. Denaturation of serum albumin by any of the methods used caused a shift in the pH optimum of albumin catabolism by tritosome extracts or by purified cathepsin D from approx. 3-4 to 5-6. These results were discussed in terms of a possible mechanism for the catabolic aspect of serum albumin turnover.     

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.     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

Design of compounds having an enhanced tumour uptake,using serum albumin as a carrier. Part I

The search for a radioiodinated “cumulative” protein label, stored within cells following intracellular protein degradation, suggested that plasma protein turnover of tumours might be of use. While earlier investigators were primarily interested in metabolism and utilization of plasma proteins by tumours, we tried to utilize the tumour protein turnover to channel radioiodine labelled compounds, covalently bound to serum albumin, into neoplastic tissues. To identify those parameters which influence the tumour uptake and storage, we investigated a series of compounds having different chemical and physicochemical properties. Unbound, small molecular weight compounds were rapidly eliminated from the circulatory system. They had a prolonged biological half life if linked to serum albumin (SA), especially when derivatized with deoxysorbitol. Parallel with the prolongation of the biological half-life we noted a remarkable increase in tumour uptake, which was not accompanied by increased liver activity. Further-more, without thyroid blockade, we failed to detect significant radioiodine uptake in this organ after 24 or 72 h. This is due to the particular coupling mechanism, which may be relevant for other (radio)iodinated pharmaceuticals used in medicine. Glucose and aromatic amines, as well as aromatic aldehydes and glucamine react to form deoxysorbitol derivates, which then have similar biokinetics after linkage to serum albumin. This indicates that a new approach in tumour detection and possibly in tumour therapy may be possible when SA is used as a carrier molecule, using the described labelling procedure.     

Hemolysis of human red blood cells by combination of riboflavin and aminophylline

The effect of aminophylline on human red blood cells (RBC) has been studied. Under in vitro condition, aminophylline alone does not hemolyse RBC. However, in the presence of riboflavin and visible light, aminophylline causes hemolysis of RBC. This hemolysis depends on the concentration of both riboflavin and aminophylline. Using different free radical scavengers we show that RBC hemolysis is caused by reactive oxygen species. Studies using bovine serum albumin show that riboflavin-aminophylline combination can also cause protein degradation in vitro.     

Retinyl esterase activity of purified rat liver retinyl ester lipoprotein complex.

Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30 °C was about fivefold larger than the activity at 4 °C, while higher temperatures than 30 °C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some 75 to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 × 10^?4 M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.