An androgen binding protein in the testis cytosol fraction of adult rats. Comparison with the androgen binding protein in the epididymis

The cytosol fraction of rat testicular homogenates contained a specific androgen binding protein (t-ABP), indistinguishable from, the androgen binding protean in. the epididymis(e-ABP) in terms of its chromatographic behaviour by gel filtration, sedimentation rate, mobility on polyacrylamide electrophoresis and steroid specificity(5α-diaydrotestosterone(DHT) > testosterone > estradiol-17β > parogesterone and estradiol-17α).The stability of t-ABP as well was similar to that of e-ABP. The aBP-DHT complexes were stable between pH 6.5–10, exhibiting the highest degree of binding between pH 8–9. The binding activity persisted after heating at 50°C for 30 min., whereas heating at 60°C for 30 min. completely destroyed the binding. DHT did not significantly increase the stability towards pH and temperature denaturation. Binding activity was not reduced by 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS), whereas similar treatment with 1 nM N-ethyl-maleimide(NEM) or 1 mM Ellman's reagent reduced it by some 10–50 per cent. Exposure to 10 mM β-mercaptoethanol(βME) reduced the binding by 60–70 per cent. These studies strongly indicate that t-ABP and e-ABP are identical proteins.Hemicastration for 4 weeks eliminated the ABP from the epididymal cytosol fraction on the castrated side, indicating that this protein is produced by the testis.     

Reversible interaction between androgen binding protein and testicular macromolecules causing inhibition of androgen binding activity.

Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.     

Afaltoxin B1 transport in rat blood plasma. Binding to albumin in vivo and in vitro and spectrofluorimetric studies into the nature of the interaction

Binding of [³H]aflatoxin B₁ to rat plasma was investigated in vivo and vn vitro. Column chromatographic and polyacrylamide gel electrophoretic analysis clearly demonstrated that aflatoxin B₁ bound primarily plasma albumin. Very little binding activity was shown by other plasma proteins. Spectrofluorimetric studies were undertaken to gain some insight into the nature of the aflatoxin-albumin interaction. Quenching of the lone tryptophan fluorescence intensity upon aflatoxin binding was due, at least in part, to a ligand-induced conformational change in the albumin molecule. Aflatoxin B₁ binds an apolar site with an association constant of 30 mM⁻¹ at pH 7.4 and 20°C. Neither charcoal treatment of rat albumin nor the presence of 0.15 M NaCl had many significant effect on the interaction. The association constant was pH-dependent, increasing about 1.7-fold as the pH increased from 6.1 to 8.4. This pH dependence is ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies indicated that the aflatoxin-albumin interaction was exothermic (ΔH = −29.3 kJ·mol⁻), with a ΔS value of −13.8 J·mol⁻¹·K⁻¹.     

Biological activity and conformational stability of the domains of plasma fibronectin

As measured by two assays of biological activity, fibronectin was readily denatured by heat. Both by the rat liver slice assay and by gelatin-latex agglutination, 90% of the activity disappeared in about 10 min at 60 °C. In contrast, immunological activity, as measured by microcomplement fixation, showed little change at 10 min and was at least 60% as great as unheated fibronectin after 20–50 min at 60 °C. Binding of heparin was unaffected by heating up to 52 min, but at very long times (48 hr at 60 °C), it also was lost. Differential scanning calorimetry of native fibronectin showed three endothermal denaturing transitions, at 68, 82, and 119 °C. Enthalpies of denaturation for the three transitions are approximately 2.6, 0.4, and 0.7 cal/g of flbronectin. These results are consistent with a three-domain structure for fibronectin. The domain which unfolds at 68 °C is associated with gelatin binding and cell. binding. The 82 °C domain appears to be associated with much of the immunological activity, and the 119 °C domain with heparin binding, as well as with some immunological activity. Residual immunological activity after loss of heparin binding may reside in nonordered portions of the molecule.     

Solubilization and characterization of an angiotensin II binding protein from liver

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II. Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high-affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high-affinity binding sites (Kd = 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure-activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non-physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68 000 by polyacrylamide gel electrophoresis.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

Production of Pentameric Hybrid Immunoglobulins Consisting of IgA and IgM

The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose.     

The effect of pH and hypertonic KC1 on the collection of the cyclic AMP-protein complex from rat skeletal muscle and erythrocyte extracts.

The effect of pH on the extent of binding of cyclic AMP was evaluated by membrane filtration, charcoal exclusion and Sephadex G-25 chromatography. The amount of binding activity found in hemolysates of rat erythrocytes and 105,000 × g supernatants of rat thigh muscle homogenates varies appreciably with pH and method of measurement. Measurements of binding activity in a muscle extract by exclusion from Sephadex G-25 indicated the presence of two pH optima, one at pH 4.5 and the other at pH 7.4 or higher. The filtration method gave higher values than the charcoal method at pH 4.5 while the reverse was true at pH 7.4. With the erythrocyte preparation no binding was evident above pH 5.0 by either procedure except in the presence of 0.8 M KCl. Hypertonic KCl raised the pH of optimum binding to 5–5.5 for both tissues as indicated by both the filtration and charcoal methods. It is apparent from these results that the determination of cyclic AMP binding proteins from various tissues requires that more attention be paid to the role of ionic strength, pH and the mode of collection of the bound complex.     

Isolation and Crystallization of Microbial Alkaline Protease Inhibitor,S-SI

S–SI, an microbial alkaline protease inhibitor, was produced in the culture broth of Streptomyces albogriseolus S–3253. S–SI was isolated from culture filtrate by salting out with ammonium sulfate, column chromatographies on DEAE-cellulose and Sephadex G–100. S–SI was also isolated by pH adjustment of the effluent from DEAE-cellulose column.

Crystallization of S–SI was carried out, and two distinct shaped crystals, needle shaped and rhombic crystal, were obtained. Homogeneity of these crystals were identified by polyacrylamide disc electrophoresis at various pHs.

S–SI retained its perfect inhibitory activity after treatment at 37°C for 25 hr in the pH range from 3 to 10, and 100°C for 10 min in the pH range from 4 to 6.

From the examination of molecular weight determination with Sephadex G–100, the molecular weight of S–SI was decided to be about 27,000.

S–SI was found to be highly specific towards microbial alkaline proteases.     

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.     

Glycogen Formation from Glycols and their Esters in the Livers of Chicks

Purification was conducted on polyvinyl alcohol (PVA) degrading enzyme produced and secreted into the culture medium by Pseudomonas O–3 strain. The enzyme was found to separate into several fractions by ion-exchange chromatography and gel filtration. Among these fractions, a fraction adsorbed to SP-Sephadex C–50 at pH 6.0 was purified to homogeneity by polyacrylamide gel electrophoresis. Some properties of this purified enzyme were examined. Optimum pH and temperature were 9.0 and 40°C, respectively. The enzyme was stable up to 50°C and in a pH range between 5 and 11 at 5°C. The enzyme activity was inhibited by Co²⁺, Ni²⁺, EDTA, hydroxylamine and salicylaldoxime. In substrate specificity, this enzyme oxidized several kinds of modified PVA, as well as normal PVA, but did not oxidize other synthetic polymers, such as vinylon, polyacrylamide and polyvinyl acetate. The effect of oxygen on this enzyme was examined, and without oxygen, PVA was not broken down by this enzyme. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G–100 to be approximately 26,000.     

Studies on the binding of a 32K rat epididymal protein to rat epididymal spermatozoa

A glycoprotein of molecular weight 32K has been isolated and purified from the rat caudal epididymal fluid by gel filtration, ion-exchange and affinity chromatography. The highly purified protein was labeled with radioactive iodine and the binding of the 125I-labeled 32K rat epididymal protein (REP) to washed rat caudal epididymal sperm was studied under various conditions. Scatchard plots of the binding data revealed two binding kinetics. One bound with high affinity (KD = 2.6 X 10(-10) ) but low capacity. The other bound with lower affinity (KD = 2.2 X 10(-9)M) but high capacity. The rate of binding of the labeled protein to sperm was dependent on the temperature of the incubation medium. At the scrotal temperature of 33 degrees C, maximal binding was obtained after 40 min. However, at 22 degrees C equilibrium state was reached after 90 min and at 0 degrees C, the equilibrium rate was not reached even after 120 min of incubation. Binding showed dependence on extracellular pH (optimal pH at 4) and ionic strength of the incubation medium. High ionic strength was found to inhibit binding of the 125I-labeled 32K REP to rat caudal epididymal sperm. Specific binding was abolished by 100-fold molar excess unlabeled 32K REP or by native rat caudal epididymal fluid proteins, but not by albumin or ovalbumin. This indicates high specificity of binding. This study has provided direct evidence for the interaction of an epididymal protein with epididymal spermatozoa.     

Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.     

pH-dependent effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor

Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37°C for 20 min, its ability to bind [³H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2–3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (>5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [³H]progesterone-receptor complexes; 70–80% of which remained after a 20 min incubation at 37°C in the presence of 2–3 mM tungstate at pH 7. At pH 8, presence of tungstate (1–10 mM) during the 37°C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4°C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.     

Purification and some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

Application of mixed modal resin for purification of a fibrinolytic enzyme

Recent advances in purification technologies for therapeutic molecules have stirred the research consortium. Mixed mode chromatography, having multiple interactions with the solute molecule, has drawn significant attention due to its overall advantage over traditional ion-exchange and reverse-phase chromatography. Capto adhere, a mixed mode chromatography resin with strong anion-exchange and reverse-phase interaction with solutes, was explored for purification of fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Static and dynamic resin binding study revealed that 30°C temperature, pH 8, and 0.5 mL/min flow rate were optimum for maximum binding of fibrinolytic enzyme. Maximum static dynamic binding and breakthrough capacities for Capto adhere were 249 and 196 U/mL of resin, respectively. Final purification with Sephadex G 100 gel chromatography resulted in 38-fold purity of fibrinolytic enzyme with 39% enzyme recovery. Purified enzyme was further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis to homogeneity, and molecular mass was found to be around 55–70 kD. Like most of the serine alkaline proteases, purified fibrinolytic enzyme was stable in a temperature range of 25–40°C and pH range of 7–9. Offshoots of our research findings have revealed a broad application area of mixed mode chromatography.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

Pathway of vesicular stomatitis virus entry leading to infection

The entry of vesicular stomatitis virus into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 °C, viruses bound to the cell surface but were not internalized. Binding was very dependent on pH. More than ten times more virus bound at pH 6.5 than at higher pH values. At the optimal pH, binding failed to reach equilibrium after more than two hours. The proportion of virus bound was irreproducible and low, relative to the binding of other enveloped viruses. Over 90% of the bound viruses were removed by proteases. When cells with pre-bound virus were warmed to 37 °C, a proportion of the bound virus became protease-resistant with a half-time of about 30 minutes. After a brief lag period, degraded viral material was released into the medium. The protease-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride blocked the infection and slightly reduced the degradation of viral protein.When the entry process was observed by electron microscopy, viruses were seen bound to the cell surface at 0 °C and, after warming at 37 °C, within coated pits, coated vesicles and larger, smooth-surfaced vesicles. No fusion of the virus with the plasma membrane was observed at pH 7.4.When pre-bound virus was incubated at a pH below 6 for 30 seconds at 37 °C, about 40 to 50% of the pre-bound virus became protease-resistant. On the basis of this result and previously published experiments (White et al., 1981), it was concluded that vesicular stomatitis virus fuses to the MDCK cell plasma membrane at low pH.These experiments suggest that vesicular stomatitis virus enters MDCK cells by endocytosis in coated pits and coated vesicles, and is transported to the lysosome where the low pH triggers a fusion reaction ultimately leading to the transfer of the genome into the cytoplasm. The entry pathway of vesicular stomatitis virus thus resembles that described earlier for both Semliki Forest virus and fowl plague virus.     

Purification and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.     

Enzymatic Properties of Alkaline Proteinase from Aspergillus candidus

Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg²⁺ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae.