Serum protein synthesis in the early chick embryo

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringer's solution. The presence of serum transferrin, two embryo-specific alpha-globulins, and a prealbumin were demonstrated by acrylamide gel analysis. Yolk-sacs from embryos explanted at 11-13 somites (40 hr preincubation) and cultured for 48 hr secreted in addition a protein with the mobility of serum albumin. Incubation of yolk-sacs in the presence of radioactive valine indicated that serum proteins were synthesized as early as the primitive streak stage. By incubating isolated yolk-sacs and embryos from 48-hr explants in the presence of radioactive valine, the synthesis of serum proteins was found to be restricted to the yolk-sac at this stage of development. Culturing explants on various nutrient proteins as well as protein starvation medium altered the relative synthesis of several serum proteins. We have proposed that morphological and biochemical changes in embryos resulting from altered nutrition may be mediated by the proteins of the serum.     

Proteins synthesized and secreted by the guinea-pig conceptus during early pregnancy in relation to corpus luteal maintenance.

Guinea-pig conceptuses obtained on Day 15 of pregnancy were cultured for 24 h in the presence of [3H]leucine. Proteins present in the culture medium were purified by dialysis, desalted, and subjected to analysis by gel filtration chromatography, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The major non-radioactive protein present co-chromatographed with serum albumin. Fresh protein synthesis occurred as indicated by the incorporation of [3H]leucine, and the proteins produced were acidic in nature. Many radioactive proteins in relatively similar amounts were produced, with protein of molecular weights 28.8 and 98.2 kDa (as determined on Sephacryl S-300HR) and of molecular weights 8.4 and 14.7 kDa (as determined on Sephadex G-75SF) being synthesized in marginally greater quantities. Consequently, from the profile of proteins synthesized and secreted, no obvious candidate emerged as the anti-luteolytic factor synthesized and secreted by the guinea-pig conceptus during early pregnancy which inhibits endometrial PGF2 alpha synthesis and maintains corpus luteal function.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

Maintenance of plasma-derived proteins at much lower concentrations in the uterine lumen of the rabbit: a kinetic study of passage.

Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Rete testis fluid (RTF) proteins: purification and characterization of RTF albumin

A major 68-kDa protein in ram rete testis fluid (RTF) is shown to be chemically and immunologically indistinguishable from albumin in ovine serum. Data obtained with two-dimensional gel electrophoresis of RTF demonstrate the presence of additional proteins with a molecular mass of 68 kDa that do not react with antisera against sheep serum albumin. Biochemical characteristics of albumin preparations isolated by immunoaffinity chromatography from ovine serum and from RTF were compared. Albumin from both sources had the same apparent molecular mass of 68 kDa, the same isoelectric point of approximately 4.2, and neither bound specifically to Concanavalin A. Analysis of tryptic peptide maps, obtained with reverse-phase high-pressure liquid chromatography, indicated no significant differences between digests of the two purified albumin preparations. Results indicate that RTF albumin and serum albumin are the same protein, which implies that RTF albumin may originate from serum. Albumin levels in RTF, collected from different rams and measured by radioimmunoassay, varied between 46 and 164 micrograms/ml, constituting between 11 and 17% of total RTF protein, while albumin levels in sheep plasma were 40,000 micrograms/ml. The protein composition of RTF is discussed in relation to the relative amounts of various components contributed by testis cells and the amounts derived from serum.     

The nature of the cytosolic activators of the adrenal steroid 21-hydroxylation system

The steroid 21-hydroxylase activity present in the microsomes of bovine adrenals is stimulated by components of the cytosol. The nature of these activators has been examined by two procedures. The first consisted of treating cytosol with increasing amounts of acetone. When the concentration of the organic solvent reached 50%, a precipitate, presumably proteinaceous, formed. The portion of the precipitate that was redissolvable in 0.05 m potassium phosphate buffer, pH 7.2, contained 7–15% of the stimulatory activity originally present in the cytosol. When the acetone concentration was raised to 90%, another active material precipitated. It was identified as oxidized glutathione (GSSG) and it accounted for about 5% of the activity in the cytosol. In an attempt to avoid the harmful effects of acetone, the second procedure employed only gel filtration and ion exchange resin chromatography. By these means the cytosol was separated into 11 protein fractions and a small molecular weight material. Forty six percent of the proteins and the same fraction of the stimulatory activity present in the original cytosol were recovered. Because all 11 protein fractions contained some stimulatory activity, the results suggested that the protein constituents of these fractions were relatively nonspecific. Yet, of the several known proteins which were tested for activity (bovine serum albumin, ovalbumin, human γ-globulin, bovine pancreatic ribonuclease, and pig insulin) only bovine serum albumin proved to be active. An additional 8% of the stimulatory activity of the cytosol was present in the fraction containing the low molecular weight components and this was all attributable to its GSSG content.     

Uptake and secretion of host proteins by Taenia crassiceps metacestodes

Although the presence of intact host proteins in the cyst fluid of cyclophyllidean metacestodes has been well documented, the underlying reason for protein uptake is poorly understood. To investigate this discrepancy, both the cyst fluid (CF) and excreted/secreted (E/S) proteins were collected in vitro from Taenia crassiceps metacestodes 16 wk postinfection in Balb/cJ female mice. The CF and E/S were subsequently immunoblotted using rabbit anti-mouse whole serum antibodies as a probe. The results show that whole host proteins were not only internalized by metacestodes but also secreted as well. The predominant secreted host protein was 66 kDa and was confirmed to be mouse serum albumin. The amount of secreted albumin decreased daily, whereas the concentration of albumin in the cyst fluid remained consistent. This suggests that the secretion of albumin is a coordinated function rather than a random event. It is probable that albumin cycling may be an evolved mechanism providing multiple benefits for the larvae, including osmoregulation and protection from innate immune responses.     

Identification of extracellular proteins in the rat cumulus oophorus

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte–cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by highresolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postvulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW ～81,000 and 100,000). The other two proteins were more basic and occurred at MW ～90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.     

A novel family of progesterone-induced, retinol-binding proteins from uterine secretions of the pig

Two-dimensional polyacrylamide gel electrophoresis has revealed the presence of a group of relatively acidic proteins of molecular weight about 22,000 in the uterine flushings of pseudopregnant pigs. The proteins have been purified by a combination of gel filtration chromatography and high performance anion-exchange chromatography and shown to bind both [3H] retinol and [3H]retinoic acid. At least four protein peaks that bound retinoids could be detected in the uterine secretions of a single pig. The ion-exchange procedure also allowed the retinol-free apoproteins to be separated from the holoforms that had associated ligand. Amino acid sequencing of the NH2 termini of polypeptides within three of the peaks revealed the presence of proteins with some degree of sequence identity to serum retinol-binding proteins (RBP). The most basic polypeptides showed the least similarity (about 30% identity), while the most acidic isoform analyzed shared about 70% sequence identity with the NH2 terminus of human serum RBP. Western blotting procedures employing an antiserum raised against the most basic isoforms showed that the amount of retinol-binding protein in uterine secretions increased markedly in ovariectomized animals in response to long term progesterone treatment. These proteins appear to form part of the uterine histotroph thought to be essential for nourishment of the conceptuses during pregnancy. A simple three-step procedure for purifying retinol-binding protein from pig serum is also described. The NH2-terminal sequence of this RBP is similar to that of human RBP but different from those of the uterine forms. The study suggests that a family of RBP, distinct from the serum form, is secreted by the uterine endometrium of the pig in response to progesterone.     

Membrane protein alterations during the early stages of sporulation of bacillus subtilis

Membrane protein alterations during the early stages of sporuloation were examined by polyacrylamide gel electrophoresis. Solubilized samples of the vegetative cell membrane (VCM), sporulation membrane fraction (SMF), and inner forespore membranes (IFM) were compared with respect to their protein compositions. The VCM contained 39 protein components, distinguishable as separate bands on gel electrophoresis, and these ranged in molecular weight from 16,000 to greater than 100,000. During the first 5 hr of sporulation, 6 of these 39 protein bands disappeared, 8 increased and 12 decreased in concentration, and 13 showed no discernible change. In addition, 15 new protein components were identified in the SMF during the fireist 5 hr. The new components consisted of 7 protein bands that were transiently associated with the SMF, and 8 proteins that persisted in the SMF from their time of appearance until at least T5 of sporulation. Comparison of the protein composition of the IFM with those of the VCM and SMF revealed that membrane protein alterations occur during sporulation. The turnover of H³-tryptophan-labeleld membrane protein was followed during growth and sporulation. During the 30 min of growth following a simple chase with excess unlabeled tryptophan, membrane protein appeared stable, whereas 5–10% of the nonmembrane protein turned over to acid-soluble material. However, manipulation of the cells by dilution ito fresh medium, or centrifugation, as part of the chase procedure, resulted in elution of membrane protein to the cytoplasm. In contrast, proteins labeled during vegetative growth were always eluted to the cytoplasm during the first 2 hr of sporulation, and this was followed by a period of reassociation with the membrane fraction. The results are discussed with respect to membrane differentiation as it relates to spore development.     

Lymphoblastoid cell-produced immunoglobulins: preparative purification from culture medium by hydroxylapatite chromatography

Hydroxylapatite (HA) is a chromatography matrix capable of separating nucleic acid as well as protein species. HA adsorbs biomolecules as a function of the extent and distribution of their surface charge. HA has been evaluated for its ability to differentially retain immunoglobulin molecules, which are planar relative to the generally globular serum proteins, particularly albumin, contained in tissue culture medium. HA chromatography provides a single-step method to purify and concentrate immunoglobulin proteins secreted by lymphoblastoid cells into culture medium from the vast excess of serum proteins used to supplement the medium. A human lambda light-chain protein was recovered in 5% of the applied volume of medium, and was separated from 95% of the total protein. More than 75% of a hybridoma-produced complete immunoglobulin was recovered essentially free of serum protein contamination. HA appears to provide a valuable alternative chromatographic medium for the purification of immunoglobulin proteins secreted by lymphoblastoid cells.     

Stimulation of protein secretion in the initial segment of the rat epididymis by fluid from the ram rete testis

Zone 1A of the ductus epididymidis was perfused with ovine rete testis fluid (nRTF) and modifications of it, and a synthetic medium (sRTF) based on the inorganic composition of nRTF. There was little fluid transport by the duct mucosa and nRTF stimulated protein secretion. The secretagogue activity was not extracted by charcoal, was sensitive to protease digestion and was present in a portion of nRTF with a molecular weight of greater than 10,000. The addition of bovine serum albumin to the sRTF stimulated protein secretion, but not to the same extent as equal amounts of protein in nRTF. Polyacrylamide gel electrophoresis of the perfusates showed that proteins with molecular weights of 19,000 (all rats studied), and 22,000, 30,000 and 60,000 (at least half the rats studied) were secreted into the perfusion fluids as well as some blood proteins, but the pattern of secretion was not affected by the composition of the perfusion fluid.     

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes (Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V-VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.     

An inhibitor(s) of globin mRNA translation in rabbit serum

1. A factor found in rabbit serum inhibits globin mRNA translation in vitro. 2. Inhibition of globin mRNA translation has been demonstrated in a cell-free rabbit reticulocyte lysate. 3. The inactivation of globin mRNA translation is not attributed to either serum albumin or ribonuclease activities. 4. Dialyzing the inhibitor for 24 hr at 4 degrees C does not result in the diminution of the inhibiting activity. However, the activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin for 2 hr. 5. Ion exchange chromatography points to the inhibitor being a neutral protein, whereas, polyacrylamide gel electrophoresis reveals one major band with mol. wt 43 kDa. 6. The activity of the inhibiting material 3-fold greater in anemic serum than in normal serum. 7. These studies suggest that rabbit serum contains a protein inhibitor that may play a physiological role in regulating protein synthesis in red cells.     

Proteins in the luminal fluid from the bovine oviduct.

Oviducal fluid was collected by cannulation from four cows and by irrigation from fifteen slaughtered cows.The proteins in the fluid were examined by polyacrylamide gel electrophoresis at pH 4-5 and pH 8-9, isoelectric focusing on polyacrylamide, immunodiffusion, immunoelectrophoresis, affinity chromatography and gel filtration. The macromolecular components found were mainly serum proteins but small amounts of other proteins were detected in oestrous and dioestrous samples by electrophoresis at pH 8-9 following fractionation of the fluid by gel filtration or affinity chromatography. Small amounts of cathodically migrating proteins were detected directly by electrophoresis at pH 4-5 in dioestrous samples but not in oestrous samples. Determination of glycosidase activities revealed that the levels at oestrus were similar to the levels detected in serum. At dioestrus, the activities of B-N-acetylgalactosaminidase and beta-N-acetylglucosaminidase were elevated.     

Evaluation of protein depletion methods for the analysis of total-, phospho- and glycoproteins in lumbar cerebrospinal fluid

A proper sample preparation, in particular, abundant protein removal is crucial in the characterization of low-abundance proteins including those harboring post-translational modifications. In human cerebrospinal fluid (CSF), approximately 80% of proteins originate from serum, and removal of major proteins is necessary to study brain-derived proteins that are present at low concentrations for successful biomarker and therapeutic target discoveries for neurological disorders. In this study, phospho- and glycoprotein specific fluorescent stains and mass spectrometry were used to map proteins from CSF on two-dimensional gel electropherograms after immunoaffinity based protein removal. Two protein removal methods were evaluated: batch mode with avian IgY antibody microbeads using spin filters and HPLC multiple affinity removal column. Six abundant proteins were removed from CSF: human serum albumin (HSA), transferrin, IgG, IgA, IgM, and fibrinogen with batch mode, and HSA, transferrin, IgG, IgA, antitrypsin, and haptoglobin with column chromatography. 2D gels were compared after staining for phospho-, glyco- and total proteins. The column format removed the major proteins more effectively and approximately 50% more spots were visualized when compared to the 2D gel of CSF without protein depletion. After protein depletion, selected phospho- and glycoprotein spots were identified using mass spectrometry in addition to some of the spots that were not visualized previously in nondepleted CSF. Fifty proteins were identified from 66 spots, and among them, 12 proteins (24%) have not been annotated in previously published 2D gels.     

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.     

Two-dimensional gel electrophoresis of endometrial protein in human uterine fluids: qualitative and quantitative analysis

By combining two-dimensional gel electrophoresis, protein staining and a sensitive computer-assisted gel scanning system, it was possible to examine human uterine fluid (n = 56) qualitatively and quantitatively for the presence of endometrial proteins. The protein concentration of uterine fluids ranged from 0.1 to 12.0 mg/ml with early secretory phase samples (n = 15) having significantly less protein (0.72 +/- 0.2 SEM mg/ml p less than or equal to 0.05) than the proliferative phase (n = 57) samples (1.58 +/- .29 SEM mg/ml). Whole blood contamination of uterine fluid, as measured by hemoglobin content, averaged 6.2 +/- 0.88% throughout the menstrual cycle. Human uterine fluids collected throughout the menstrual cycle were found to contain serum and up to 24 other proteins in addition to those previously described (MacLaughlin and Richardson, 1983). These proteins represent approximately 1% of the total protein in the gels and exhibit isoelectric points from 4.5 to 7.0 and molecular weights in the 26,000 to 60,000 range. These proteins are absent from human serum, which exhibits an identical pattern whether obtained in the proliferative or secretory phase of the menstrual cycle. These secreted endometrial proteins now become the standard against which to compare proteins identified in vitro using organ, gland and cell culture techniques and to characterize proteins that are regulated by steroid hormones in vivo.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.