Isolation of porcine follicular fluid inhibin of 32K daltons

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.     

On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA

DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.     

Effects of cysteine to serine substitutions in the two inter-chain disulfide bonds of insulin

Using site-directed mutagenesis we deleted the two inter-chain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two inter-chain disulfide bonds resulted in the complete loss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico-chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptor-binding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversed-phase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.     

Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture

The viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs-Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose-dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1-x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE-2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.     

Presence of bacteria in porcine follicular fluid and their ability to generate an inhibitor of follicle-stimulating hormone binding to receptor

Follicular fluid obtained from porcine ovaries collected at slaughter and distributed by the National Institutes of Health was contaminated by bacteria which appeared to be of intestinal origin. This follicular fluid showed increased follicle-stimulating hormone binding inhibition (FSH-BI) activity following incubation under conditions which facilitated bacterial growth. No such increase in FSH-BI activity was observed following incubation of follicular fluid from which bacteria were removed by repeated filtration. Our data suggest that bacteria found in the follicular fluid were capable of generating a substance with FSH-BI activity. This substance has an apparent molecular weight greater than 6000, based on membrane diafiltration studies. The possible presence of bacteria in follicular fluid and their ability to generate a substance which interferes with FSH binding to receptor should be considered in studies on factors in follicular fluid that are considered to regulate ovarian function or development.     

Identification, purification and immunohistochemical detection of the inhibitor from porcine ovarian follicular fluid to compensatory ovarian hypertrophy in mice.

An inhibitor to compensatory ovarian hypertrophy in mice was detected in porcine follicular fluid. Compensatory hypertrophy was inhibited by treatment with 0.6 ml whole follicular fluid, 0.2 ml of the non-dialysable fraction, or a fraction of the fluid salted out by ammonium sulphate at a saturation of 14.5--18.5%. The salted-out fraction was separable into two peaks by Sephadex G-200 column chromatography and the second peak, detectable as a single band by polyacrylamide gel disc electrophoresis, contained all the inhibitory activity. Specific fluorescence of an antiserum to the second peak was demonstrated at the granulosa cells.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.     

Characterization of porcine plasma fibronectin and its fragmentation by porcine liver cathepsin B

Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid

Inhibin from bovine follicular fluid was partly purified using affinity chromatography on immobilized Procion Red 3B, gel filtration on Sephadex G-25 and ion-exchange chromatography on the fast protein liquid chromatography system. Inhibin was subsequently characterized using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. Biological activity was associated with a protein with an apparent molecular weight of approximately 65 kD.     

Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis

Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.     

Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering.

Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.     

Interchain disulfide bonds promote protein cross-linking during protein folding

We provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH < 7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide cross-links; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.     

Oxidative refolding chromatography: folding of the scorpion toxin Cn5

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.     

Purification of follicular regulatory protein: possible plasminogen identity

A partially purified protein (the SR fraction) of porcine and human origin has been extensively characterized as Follicular Regulatory Protein (FRP). In the current study, 1A8D5, one of several monoclonal antibodies raised against FRP, was used to further purify the protein. The monoclonal antibody cross-reacted only with porcine plasminogen, a key fibrinolytic proenzyme. A commercial polyclonal antibody for human plasminogen confirmed the relationship between plasminogen and bands of the SR fraction of the porcine follicular fluid. Sequencing of the N-terminal amino acids (54 kd) of the SR fraction indicated that it shared 100% identity with the short form of porcine plasminogen chain A and 93% identity to human plasminogen. Moreover, we demonstrated that this purified protein from human follicular fluid inhibited aromatase activity of granulosa cells, a key biological property of FRP. Given that plasminogen possesses most of the proposed properties of the protein termed FRP, we conclude that FRP is likely plasminogen itself or a plasminogen-related protein and not a novel protein.     

Purification and partial characterization of inhibin from porcine follicular fluid

Inhibin, a protein of gonadal origin that suppresses the basal secretion of follicle stimulating hormone by anterior pituitary cells has been purified from porcine follicular fluid. Using several RP-HPLC steps and gel filtration under denaturing conditions, we obtained a fraction approximately ten thousand fold purified which showed one band on SDS PAGE and in the same experiment two bands after reduction (MW ca 14K and ca 18K) suggesting a molecular weight of 32K for inhibin. Edman degradation of isolated inhibin and carboxymethylated chain A indicated that the first 6 residues were H-Ser-Thr-Ala-Pro-Leu-Pro-; by subtraction, the first 3 residues of chain B could be deduced to be H-Gly-Leu-Glu-. EC50 was ca 0.3 ng/ml or 10 pM in our in vitro pituitary cell culture assay. Antibodies to residues 1-6 were raised which could immunoneutralize purified inhibin activity in an in vitro assay.     

Oxidative Inhibition of Protein Phosphatase 2A Activity: Role of Catalytic Subunit Disulfides

A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A_C) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A_C. Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A_C vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol–disulfide equilibrium of a vicinal thiol pair of PP2A_C may confer redox sensitivity on cellular PP2A.     

Purification of human lymphoblastoid interferon by a simple procedure with high yields

Human Namalwa cell interferon, induced by Sendai virus and composed of a single species with molecular weight of 17,000, was purified to 4.5 X 10(8) international reference units/mg of protein by a combination of salt precipitation, ion exchange chromatography, metal chelate chromatography, hydrophobic chromatography, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. By immunization of a rabbit with this purified interferon and by extensive absorption with Namalwa cells and an impurity column, highly specific antibody was obtained. Namalwa cells, treated with 5-bromo-2'-deoxyuridine, produced 10-fold more interferon upon induction by Sendai virus. Interferon in this case consisted of heterogeneous species with molecular weight ranging from 15,000 to 24,000. These heterogeneous interferon molecules were purified to 7.6 X 10(8) international reference units/mg of protein by successive chromatography using immobilized highly specific rabbit anti-interferon antibody, Blue Sepharose, and immobilized goat anti-rabbit IgG antibody. The overall recovery of interferon activity was 72%, and the purity of the final preparation was ascertained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.