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1.
Abstract

The widespread use of bovine serum albumin preparations for the stabilization of purified glycosyltransferases has prompted us to study the effects of different preparations of albumins on the galactosyltransferase activity of bovine milk. For comparison, several other proteins were tested as well. The albumins caused a large stimulation of transferase activity (400–700%) which varied depending on the source of the albumin and the treatment to which it had been subjected. Several other unrelated proteins were tested for their effects on transferase activity. Some proteins stimulated, while others had little effect. Lysozyme stimulated the activity by 178% and poly-L-lysine had Vittle effect. Other proteins stimulated to variable extents. The stimulations obtained with albumin and myelin basic protein were noteworthy. The stimulation was considerably less marked when the enzyme was incorporated into lipid vesicles.  相似文献   

2.
1. The influence of bovine serum albumin and soluble rat liver proteins on the activity of rat liver microsomal delta9 and delta6 desaturases has been studied. 2. In the absence of bovine serum albumin, the delta9 desaturase which converts stearoyl-CoA into oleoyl-CoA, shows a non-linear correlation between enzyme activity and protein concentration. 3. Optimum concentrations of bovine serum albumin have three main effects on the enzyme activity: (i) establishes a linear relationship between enzyme activity and protein concentration, (ii) stimulates the enzyme activity 2--3-fold and (iii) raises the optimum substrate concentration from 10 to 100 muM. 4. A highly purified soluble liver protein of molecular weight 24 000 also stimulated the enzyme activity and brought about a linear relationship between enzyme activity and protein concentration. 5. It was concluded that the non-linear kinetics were due to limiting amounts of substrate binding protein in the microsomal preparations. 6. The delta6 desaturase which converts linoleoyl-CoA into gamma-linolenoyl-CoA was also stimulated by bovine serum albumin and soluble liver proteins. 7. The significance of the fatty acid-binding proteins is discussed.  相似文献   

3.
Summary Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted, in the isolation of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors. The work was supported by grants MT-6072 and ME-9031 from the Medical Research Council of Canada. The author is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Quebec.  相似文献   

4.
Serum albumin was an effective as whole serum or α-globulins in facilitatting sterol release from strain L mouse fibroblasts. Commercial bovine serum albumin preparations, however, had markedly different absolute effects in this regard. These differences were attributable to the variation in phospholipid content of these products. All but one of these albumins enhanced sterol release when supplemented with phospholipid. The exception was fatty acid-poor albumin which contained an adequate amount of phospholipid. Among the phospholipids examine, lecithin proved to be most effective, while phosphatidylethanolamine had little potentiating influence. As the unsaturation of the test lecithins increased, enhancement of sterol release decreased. The potentiating effect of the phospholipid was in turn dependent on the protein used, since the phenomenon was not observed with non-serum proteins like ovalbumin or with non-transport serum proteins such as γ-globulins. The results of these studies raise the possibility that serum albumin together with phospholipid can play an important role in sterol release in tissue cultured cells.  相似文献   

5.
Various proteins modulate the kinase activity of the insulin receptor   总被引:2,自引:0,他引:2  
Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.  相似文献   

6.
Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.  相似文献   

7.
The Δ5-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ5-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.  相似文献   

8.
Inhibition of soluble guanylate cyclase by bovine serum albumin   总被引:1,自引:0,他引:1  
Bovine serum albumin (BSA) and to a lesser extent beta-lactoglobulin produced concentration-dependent inhibition of the guanylate cyclase activity in supernatant fraction and partially purified enzyme (PPE) prepared from rat lung homogenates. Ovalbumin had little effect. Some activity was lost when PPE was applied to a BSA-agarose column, however the loss disappeared when the enzyme reaction mixture contained Lubrol PX. Also, BSA no longer inhibited PPE after BSA-agarose treatment. BSA inhibition of PPE was not apparent when activity was maximally stimulated by arachidonate. These data were interpreted as indicating that the enzyme had bound to it an amphiphilic activator, possibly a fatty acid, the removal of which by BSA decreased activity.  相似文献   

9.
Summary The protective influence of bovine serum albumin against growth inhibition caused by fatty acids was studied in human hepatoma (HepG2) and immortalized human kidney epithelial (IHKE) cells. In general, growth inhibition by unsaturated fatty acids (0.15 mmol/liter) increased with increasing number of double bonds. For HepG2 cells crude albumin (1g/100 ml) did not greatly modify growth inhibition by arachidonic, eicosapentaenoic, and docosahexaenoic acid. With oleic, linoleic, and linolenic acids, crude and defatted albumin stimulated cell growth. In contrast, for IHKE cells both albumins counteracted growth inhibition by unsaturated fatty acids to approximately the same extent. When HepG2 cells were cultured in the presence of saturated fatty acids (0.3 mmol/liter), C2, C6, and C8 had no or little inhibitory effect. C10 and C12 inhibited cell growth appreciably, whereas C14, and especially C16, had poor inhibitory effects. Crude albumin counteracted growth inhibition by all these fatty acids. In contrast, defatted albumin had little or no effect (except against C10 and C12), and even increased the growth inhibition by C14 and C16. With unsaturated fatty acids there seemed to be an inverse relationship between cell growth and the concentration of thiobarbituric acid reactive substances (TBARS) in media. Vitamin E abolished growth inhibition (and the increase in TBARS concentration) by unsaturated fatty acids. The complex interaction between fatty acids and albumins calls for great caution when interpreting data on growth effects.  相似文献   

10.
Epidermal growth factor, a potent mitrogen for granulosa cells produced a three-fold stimulation of ornithine decarboxylase activity in porcine granulose cells in vitro. Fibroblast growth factor, another compound with mitogenic activity for granulose cells, did not stimulate ornithine decarboxylase. Maximally effective concentrations of a commercial preparation of bovine serum albumin equalled the maximal effect of epidermal growth factor on this enzyme activity. The dominant stimulator(s) in the albumin preparation eluted after bovine serum albumin in gel filtration. At maximally effective concentrations, luteinizing hormone produced substantially greater stimulation than either epidermal growth factor or the bovine albumin preparation. Combinations of saturating doses of any two of these stimulators produced additive effects on enzyme activity.  相似文献   

11.
Nine styrylpyridine analogs were tested as inhibitors of choline acetyltransferase which had been highly purified from rat cerebrum and bovine caudate nuclei. In general, concentrations required to achieve 50% inhibiion (I50 values) were in the micromolar range. For some analogs, I50 values were similar to those obtained previously by other investigators who used less purified enzyme preparations. With certain analogs, however, the measured values of I50 changed as the transferase became more purified, which may indicate the presence in the extract of other molecules which can interact with the enzyme. The methods used in purification of the enzyme suggest that the molecule which modifies the activity of CAT is probably a protein. The mode of inhibition by naphthylvinylpyridinium was found to be uncompetitive with respect to both choline and acetyl coenzyme A for both the rat and bovine transferases.  相似文献   

12.
When bovine serum fraction V powder, which contains 98-99% albumin, is incubated with rat liver microsomes, the glutathione S-transferase activity is stimulated 3-fold. The fraction V stimulates the transferase activity even in microsomes pretreated with N-ethylmaleimide. There is no such stimulating activity in protein fractions other than fraction V. The corresponding cytosolic enzyme activity is not changed by fraction V powder or by N-ethylmaleimide. Neither treatment with charcoal nor heating in boiling water for 5 min can remove the stimulating activity from fraction V.  相似文献   

13.
Human neutrophils contain a neutral metalloproteinase which degrades denatured collagens and potentiates the action of interstitial collagenase. This gelatinase is rapidly secreted from neutrophils stimulated with phorbol myristate acetate. The secreted enzyme has been purified by a combination of chromatography on DEAE-cellulose and gelatin-Sepharose. The purified enzyme was latent and had a specific activity of 24,000 units. Estimated molecular weight obtained by gel filtration was 150,000-180,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed three bands with relative molecular weights of 225,000, 130,000, and 92,000. Electrophoresis in the presence of a reducing agent revealed a single band of Mr = 92,000. All the proteins seen on the unreduced gel were found to contain proteolytic activity against gelatin and native type V collagen. Polyclonal antibodies were prepared against the Mr = 130,000 and 92,000 proteins. When analyzed by immunoblotting, both antibodies recognized all three proteins. Furthermore, the identical three proteins were identified by the antibodies when crude culture medium was immunoblotted. The purified enzyme was inhibited by EDTA and 1,10-phenanthroline but not by serine or thiol proteinase inhibitors, suggesting that the enzyme is a metalloendoproteinase. The enzyme had little or no activity against common protein substrates such as bovine serum albumin or casein. Native type I collagen was not cleaved under conditions where native type V collagen was extensively degraded.  相似文献   

14.
Purified bovine milk galactosyltransferase was combined with liposomes of different lipid composition. The activity was markedly affected by the nature of the lipid used. Thus phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol stimulated the activity, while phosphatidic acid and phosphatidylserine inhibited the activity of the transferase. Phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid had identical fatty acid compositions, yet phosphatidylcholine and phosphatidylglycerol stimulated the activity while phosphatidic acid inhibited the activity. The effect on the enzyme was probably related to the nature of the head group since the inhibition by phosphatidic acid could be converted to stimulation by methylating the phosphatidic acid. The properties of several of the head groups is discussed. The physical state of the lipid was shown to affect the activity markedly. When the enzyme was combined with dimyristylphosphatidylcholine the activity was markedly stimulated when the lipid was in the liquid-crystalline state i.e., above the phase transition.  相似文献   

15.
Xanthine oxidase, isolated from bovine milk, exhibited an A280:A450 nm ratio of 5.0. This ratio is reported to be indicative of highly purified enzyme preparations. Serum from a rabbit hyperimmunized against this enzyme fraction exhibited two precipitation lines when incubated with the protein in agarose double diffusion plates. Serum albumin, beta-lactoglobulin, alpha-lactalbumin, lactoferrin, casein, chymosin, and immunoglobulin were tested for reactivity. The second antigen was identified as bovine immunoglobulin. Commercial preparations of xanthine oxidase also contained immunoglobulin as a contaminant. IgG and IgA were present in Sigma (Grade III) fractions and IgM was identified in Boehringer Mannheim preparations. Immunofluorescent studies indicated that xanthine oxidase antiserum reacted with the capillary endothelium of bovine heart. Absorption of this antiserum with bovine IgG abrogated this reaction. These findings may explain apparent discrepancies between reported immunohistological association of xanthine oxidase in heart capillary endothelial cells and the absence of detectable enzymatic activity.  相似文献   

16.
Crystalline bovine serum albumin increased the mechanical resistance of fresh human erythrocytes to lysis by hydrodynamic shear forces. A saturation effect suggests that the bovine alubmin molecules are adsorbed on to a finite number of “attachment sites” on the erythrocyte surface, possibly by displacing human proteins already occupying these sites. A heterogeneous fraction of human serum albumins does not exhibit the same marked protection effect, nor displace adsorbed bovine albumin molecules from the erythrocyte surface. The precise nature and extent of the interaction between any given concentration of either human or bovine serum albumin and the intact erythrocyte membrane depends upon the chronological age of the cell concerned.  相似文献   

17.
The activity of bull sperm hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36) is increased by the inclusion of polycations in the assay mixture. At pH 3.8, bovine serum albumin and histone give the greatest stimulation, while protamine sulfate, spermine, spermidine and hyamine 2389 stimulate to a lesser extent. Enzyme activity increases with serum albumin concentration to a nearly constant, high level at serum albumin concentrations greater than 1 mg/ml. Other stimulatory compounds show a similar concentration dependence except that inhibition of enzyme activity occurs at high concentrations of stimulator. The degree of stimulation depends on the pH, sample concentration and substrate concentration. Enzyme preparations with a low protein content give the greatest stimulation, while preparations with a high protein content show little stimulation. The concentration of serum albumin required for maximum stimulation increases with increased hyaluronic acid concentration. The results suggest that the stimulation of sperm hyaluronidase is nonspecific and results from an interaction of the polycation with hyaluronic acid. Since protein in the enzyme preparation substitutes for exogenous stimulator to a varying degree, serum albumin should be included in the assay mixture for sperm and testicular hyaluronidase to assure measurement of maximum enzyme activity.  相似文献   

18.
Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.  相似文献   

19.
The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.  相似文献   

20.
The activity of purified bull seminal RNase was markedly stimulated by various basic proteins. At the half concentration of substrate RNA, basic proteins such as histones, high-mobility group chromosomal proteins and cytochrome c stimulated the enzyme activity 4-6 fold. Other non-basic proteins such as bovine serum albumin and human gamma-globulin were far less effective. In addition to enzyme-stimulating activity, basic proteins showed a marked enzyme-stabilizing activity, indicating the presence of a strong interaction between the enzyme and basic proteins.  相似文献   

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