Studies on the different forms of material reacting with antiinsulin antibodies in the fetal and adult rat.

The nature of peak B (MW = 10-12000, proinsulin?) and PEak C (MW = 50-100000, "big big" insulin?) materials detected by the double antibody (DA) procedure in elution profiles of rat sera after Sephadex G 50 or G 100 chromatography (cf. preceding companion paper) is further investigated. Peak B is converted by mild tryptic digestion in an immunoreactive material behaving in rechromatography exactly like insulin monomer. Peak C is less easily detected by the dextran coated charcoal (DCC) method; it resists 8 M urea 37 degrees C for 1 hr, is not an artifact due to the complement system; its relative importance is very much reduced in pancreatic extracts or perifusates. Incubation of biologically active 125I labelled insulin in rat sera results in appearance of labelled material behaving on chromatography like peak C natural material, having the electrophoretic mobility of rat alpha I globulins and albumin, and resisting 8 M urea, acidic pHs and 0.5 M NaCl. Similar incubation in buffer supplemented with bovine albumin results in appearance of a labelled material having the electrophoretic mobility of beef albumin; N-ethyl-maleimide provides against this binding, which might result from (S-S)-(SH) interchanges. Rat alpha globulins and albumin (but not beef albumin) cross-react with the DA procedures; they do not react with the DCC method. Insulin bound to plasma proteins reacts with both methods. It is suggested that peak C material, as detected by the DA method in rat serum, consists both of insulin covalently bound to plasma proteins and of certain plasma proteins; the DCC method detects only bound insulin. In streptozotocin treated rats, peak C material persists after the complete disappearance of insulin and proinsulin, when detected by the (DA) procedure, but disappears when detected by the DCC procedure.     

The reactions of hemopexin and liver fatty acid-binding protein (L-FABP) with aurothiomalate

We investigated whether gold(I) binds to hemopexin, Hx, a serum heme carrier with a molecular weight similar to that of serum albumin, or to L-FABP, a liver cytosolic heme- and fatty acid-binding protein whose molecular weight is similar to that of metallothionein. These proteins and, for comparison, similar concentrations of bovine albumin were incubated with sodium aurothiomalate and then fractionated by gel exclusion chromatography. At 16 μM of Hx and gold the ratio of gold bound to Hx was 0.30 ± 0.04. The corresponding ratio for albumin was 0.98 ± 0.04 per mercaptalbumin. Considering the much lower serum levels of Hx compared to albumin it is unlikely that Hx plays a role in serum gold binding and transport. L-FABP also binds gold: at 52 μM L-FABP and 108 μM gold, the gold to protein ratio was 0.55 ± 0.05. The corresponding ratio for albumin under identical conditions was 1.18 ± 0.08 per mercaptalbumin. L-FABP failed to bind zinc or cadmium, two other metals bound by metallothionein.     

Comparison of biotin binding protein of pregnant rat serum with rat serum albumin

The purified biotin binding protein of pregnant rat serum was shown to be immunologically similar to rat serum albumin as assessed by a sensitive radioimmunoassay. In radioimmunoassay for rat biotin binding protein, the binding of [¹²⁵I] rat biotin binding protein to anti-chicken egg yolk biotin binding protein antibodies was displaced by both rat serum (10–100 nl) and purified rat serum albumin (0.1–10 ng). Similarly, in radioimmunoassay for rat serum albumin the binding of [¹²⁵I] rat serum albumin to either anti-rat serum albumin antibodies or anti-chicken egg yolk biotin binding protein antibodies was displaced by unlabelled rat biotin binding protein at comparable concentration range (0·5–10 ng). Significant fractions of radioiodinated rat biotin binding protein and rat serum albumin bound to antibodies to chicken egg yolk biotin binding protein. In immature rats, the circulating half-lives of rat biotin binding protein and rat serum albumin were determined to be 12 and 17 h respectively. The rat biotin binding protein and rat serum albumin were analysed by techniques that exploit their physicochemical properties. They displayed similar electrophoretic mobilities in alkaline as well as denaturing sodium dodecyl sulphate-polyacrylamide gels. However, in nonequilibrium pH gradient polyacrylamide gel electrophoresis, they resolved clearly. In two-dimensional tryptic peptide map analysis, the two proteins showed similarities as well as significant differences in the relative distribution patterns of their iodopeptides. These results showed that the primary structure of rat biotin binding protein and rat serum albumin were different in finer details despite the fact that they shared significant immunological cross-reactivity.     

Demonstration and ontogenesis in the rat of a serum protein analogous to human thyroxine binding globulin

It has been reported evidence based on equilibrium binding, electrophoretic, immunoelectrophoretic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). It is shown that in the sera of postnatal developing animals, between 3 and 21 days, the thyroxine (T4) and the triiodothyronine (T3) binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as T4 carrier to the thyroid binding prealbumin (TBPA), but retains the major role as binder of T3, i.e. of the biologically active thyroid hormone.     

Plasma diethylstilboestrol binding proteins of rat, mouse and man in the course of development: relations with the binding of estradiol.

High diethylstilboestrol (DES) binding has been demonstrated in fetal and adult sera from man, rat and mouse by equilibrium dialysis and electrophoretic techniques. In the adults of the three species and in the human fetus only albumin shows an elevated binding capacity for DES. By contrast, in the case of rat and mouse embryos there are two proteins, namely albumin and alpha-fetoprotein, which afford major and quantatively similar contributions to the binding. Human alpha-fetoprotein does not bind DES. These phenomena are analysed in relation to the estrogen binding characteristics of the alpha-fetoproteins of the three species.     

The binding of silica to proteins from plasma and lungs of rat: in vitro

Silica dissolving out from the slate dust was found to bind with plasma protein and purified bovine serum albumin. At 24 h of incubation at 37 degrees C binding affinity of silica (microgram of silica bound/mg of protein) with plasma protein and bovine serum albumin was found to be 0.59 and 0.44, respectively. By molecular exclusion chromatography using Sephadex G-200, silica binding protein of plasma was determined to be of mol. wt. around 67000. Similar proteins having silica binding capacity (mol. wt. 70000 and 85000) were also found in rat lung but these proteins unlike their plasma counterpart were glycoprotein in nature. Polyacrylamide gel electrophoresis of plasma and protein rich lung fraction show that proteins upon binding with silica undergo mobility changes. Significance of the existence of silica binding protein in plasma and lung of rat in relation to silica toxicity is discussed.     

Affinity of uterine luminal proteins for rat blastocysts.

The binding of 125I-labelled rat uterine luminal proteins from Day-5 pregnant rats showed higher binding affinity to blastocysts than did the binding of proteins in uterine fluid from pro-oestrous rats (Day 0), rat serum albumin (RSA) or bovine serum albumin (BSA). Apparently little uptake of proteins into cells by phagocytosis or entry into the blastocoelic cavity occurred since similar results were obtained in the presence of sodium azide or cytochalasin B. Autoradiographic studies showed that the proteins were localized on the outer surface of the blastocyst. The binding was Ca2+-dependent. Denaturation of Day-5 uterine proteins at 80 degrees C reduced the counts to the values obtained with undenatured RSA and Day-0 fluids; this residual binding was considered as non-specific. The binding of labelled Day-5 uterine proteins was substantially reduced in the presence of unlabelled Day-5 proteins but to a lesser extent in the presence of RSA or rat serum. The dissociation of the bound labelled Day-5 uterine proteins occurred most rapidly in the presence of unlabelled Day-5 proteins. However, dissociation occurred within 2 h in the presence of other macromolecules, suggesting that the binding was not strong.     

Alteration in brain proteins following occlusion of the middle cerebral artery in rat.

The effects of permanent focal ischemia on specific proteins of the cerebral hemisphere were studied by unilateral occlusion of the middle cerebral artery (MCAO) in rat. Brain proteins were prepared 72 h after the occlusion and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were identified by their interaction with rabbit antibodies against rat serum proteins and anti-transferrin antibodies. SDS-PAGE analysis of the proteins prepared from ischemic tissue showed significant increase in the 66 and 80 kDa components; where a marked decrease in the 260 kDa protein occurred in the ischemic and para-ischemic tissues. The 66 kDa and 80 kDa proteins stained intensely with anti-serum protein antibodies, indicating that they are related to plasma components. Moreover, the 66 kDa band had the same electrophoretic mobility as bovine serum albumin used as a standard molecular size marker. The 80 kDa band was identified as transferrin by staining with the specific antibody. Transferrin was immunolocalized in the penumbra of cerebral cortex, hippocampal CA1 region and dentate gyrus of the ischemic cerebral hemisphere. The present results suggest that alteration in the brain content of 66 kDa (albumin), 80 kDa (transferrin) and 260 kDa (unidentified) proteins may reflect early effects of focal ischemia.     

Evaluation of safety and efficacy of a gold containing Ayurvedic drug

Gold containing Ayurvedic preparation, Swarna Vasant Malti, was given to 20 male persons in a dose of 100 mg twice a day for 40 days under supervision of Ayurvedic physicians. The total cumulative intake of 160 mg of gold at the rate of 4 mg per day in this form did not have any toxic effect on human body as evidenced by clinical examination, unaltered body weight, absence of urinary pathology and by 30 sensitive biochemical and enzymatic tests. The gold from this Ayurvedic preparation was found in plasma and erythrocytes, excreted partly in urine and was present in semen. Gold binding to albumin and hemoglobin slightly increased their electrophoretic mobility towards anode. This gold preparation seemed to increase sperm motility and prostatic activity.     

A thyroxine binding globulin (TBG)-like protein in the sera of developing and adult rats

We report evidence based on equilibrium binding, electrophoretic, autoradiographic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). We show that in the sera of postnatal developing animals, the thyroxine and the triiodothyronine binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as thyroxine carrier to the thyroid binding prealbumin, but retains the major role as binder of triiodothyronine i.e. of the biologically active thyroid hormone.     

Characteristics of aldosterone binding in rat and human serum

Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.     

Participation of the lone tryptophan residue of rat alpha-foetoprotein in its drug-binding sites. Comparison with rat serum albumin.

The participation in drug binding of the lone tryptophan residue of rat alpha-foetoprotein (alpha-FP) and serum albumin, the two main transport proteins of foetal serum, has been studied by two different techniques. Firstly, the effect on phenylbutazone and warfarin binding of the chemical derivatization of the lone tryptophan residue of both proteins by 2-nitrophenylsulphonyl chloride (NPS) was studied. Secondly, the effect of phenylbutazone binding on the intrinsic fluorescence of the tryptophan residue of rat alpha-FP and albumin was investigated. The specific modification of the proteins by NPS did not affect the binding of warfarin by rat alpha-FP and albumin, but greatly decreased the affinity of the high-affinity sites of rat alpha-FP for phenylbutazone, though the numbers of these sites were not significantly changed. However, for albumin a similar decrease in the affinity constant appeared to be due to the reaction conditions. The spectrofluorimetric studies showed that the lone tryptophan residue of alpha-FP and albumin was quenched by phenylbutazone binding, and the quenching paralleled the fractional saturation of the high-affinity site only in the case of albumin. The effect of phenylbutazone binding on the intrinsic fluorescence of rat alpha-FP indicated that the lone tryptophan residue of this foetal protein is not in the same molecular environment as that of albumin, not participating directly in the high-affinity site for phenylbutazone, and the effect may be via some induced conformational change in rat alpha-FP. These results also confirm our previous suggestion that the high-affinity sites for phenylbutazone and warfarin are different on the rat alpha-FP molecule. The results seem to indicate that this is also the case for albumin, but confirmation is necessary.     

Differences in the binding of thyroid hormones and indoles by rat alpha 1-fetoprotein and serum albumin

The transport of small molecules in the blood, normally assured by serum albumin in the adult, is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the alpha 1-fetoprotein concentration. In order to investigate whether rat alpha 1-fetoprotein might be a fetal counterpart to albumin, the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues. Though high-affinity binding of thyroxine was found with both rat alpha 1-fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two proteins. Further, while rat serum albumin strongly bound L-tryptophan and indolyl-3-acetic acid (Ka approximately equal to 10(5) M-1), rat alpha 1-fetoprotein did not bind any of the indoles tested. These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

LABELING WITH 14C AMINO ACIDS OF ALBUMIN-LIKE PROTEIN BY RAT LIVER RIBONUCLEOPROTEIN PARTICLES

Ribonucleoprotein particles were prepared by treatment of rat liver microsomes with detergents and high concentrations of KCl. They were active in incorporating ¹⁴C amino acids into protein when incubated with cell sap together with ATP, GTP, and a system to regenerate the triphosphates. The albumin of the incubation mixture, soluble at 105,000 g, and that of the fraction released by ultrasonication of the particles were studied by immunoelectrophoresis in agar gel. When the ribonucleoprotein particles were incubated with cell sap the immunological precipitation lines formed with antiserum to rat serum albumin were highly radioactive as tested by autoradiography. After zone electrophoresis on cellulose acetate, two immunologically reactive albumins were obtained which differed in their electrophoretic mobility from rat serum albumin. Labeled albumin, when purified on DEAE-cellulose columns, retained its radioactivity as tested by autoradiography following immunoelectrophoresis. On cellulose acetate this purified albumin showed an electrophoretic mobility higher than that of rat serum albumin.     

The distribution of rat testibumin in the male reproductive tract

A glycoprotein, designated CMB-1, has been identified in media from Sertoli cell-enriched cultures that increases in concentration in response to follicle-stimulating hormone (FSH) and testosterone. Subsequent studies indicated that CMB-1 is immunologically related to albumin and alpha-fetoprotein and is concentrated in the luminal compartment of the testis in adult rats. Thus, CMB-1 was termed testibumin. The goal of the present study was to determine the concentrations of this protein in testes, epididymides, and serum of normal rats between 10 and 180 days of age and to compare them to rat androgen-binding protein (rABP). Testibumin concentration in rat testes increased with age and peaked at Day 60; thereafter, unlike rABP, its concentration declined, reaching a plateau by 150 days of age. Testibumin concentration in the epididymal compartment also increased with age and peaked at Day 90; thereafter, its concentration remained relatively unchanged. Unlike rABP, which accumulates in the caput epididymis, testibumin did not accumulate preferentially in any particular region of the epididymis. In spite of the marked changes of testibumin concentration in the male reproductive tract, the levels in blood remained relatively constant between 10 and 180 days of age. In adult male and female rats, the serum concentrations of testibumin were similar. Following orchiectomy, serum testibumin concentration decreased by 50% with an apparent t1/2 of approximately 8 h. The presence of immunoreactive macromolecules in other species that share epitopes with rat testibumin was also investigated. Material in human sera and extracts of human and monkey testes cross-reacts with rat testibumin. After [35S]methionine was added to the primary Sertoli cell-enriched cultures, anti-testibumin antiserum selectively immunoprecipitated a radiolabeled protein with the same electrophoretic mobility as purified testibumin on sodium dodecyl sulfate (SDS)-polyacrylamide gels. We conclude that 1) rat testibumin is synthesized and secreted by Sertoli cell-enriched cultures; 2) the relative concentrations and distribution of testibumin in testis, epididymis, and serum of the rat as a function of age are strikingly different from those of rABP; 3) rat testibumin shares epitopes with proteins in human serum and testicular extracts of monkey and man.     

Binding of physostigmine to rat and human plasma and crystalline serum albumins

The binding of 3H-physostigmine (3H-Ph) to human and rat plasma proteins and crystalline serum albumin was studied by ultrafiltration technique. This study showed that the percentage of 3H-Ph bound to rat plasma slightly decreased from 49% to 41% whereas human plasma showed an increase in binding from 29% to 43% over a 50-fold increase in drug concentration. Human plasma samples which were collected in a bag coated with citrate phosphate dextrose adenine-1 solution bound 50% less 3H-Ph than samples collected with EDTA indicating a drug-drug interaction between 3H-Ph and anticoagulants. No significant change in binding was observed if the samples were frozen prior to use. Scatchard plots for binding of 3H-Ph resulted in a positive slope for human plasma and a negative slope for rat plasma; whereas curvilinear Scatchard plots with negative slopes were obtained for binding to human and rat crystalline serum albumin.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

An assay for albumin messenger RNA in an vitro protein synthesizing system from wheat germ.

The synthesis of serum albumin in an in vitro protein-synthesizing system from wheat germ stimulated with rat liver polysomal RNA is demonstrated by immunoprecipitation. The newly synthesized albumin has the same electrophoretic mobility as rat serum albumin. There is a linear increase in precursor incorporation into total protein and albumin with increasing RNA concentration. Potassium and magnesium optima for albumin synthesis are different from those for total protein synthesis.