Isolation and partial characterization of intestinal calcium-binding proteins from the cow, pig, horse, guinea pig, and chick.

1. Intestinal calcium-binding proteins have been isolated in high purity from mucosal tissue of the cow, pig, horse, guinea pig, and chick. The proteins from all species exhibit rapid, although not identical, electrophoretic mobilities and possesses high affinities for calcium. 2. The intestinal calcium-binding proteins of mammalian origin exhibit a molecular size of approx. 11 000 by calibrated gel filtration and 9000 on the basis of amino acid composition. The analogous chick protein was found to be about 27 000-28 000 molecular weight by these methods. 3. The amino acid composition of each intestinal calcium-binding protein has been determined and indicates a considerable degree of similarity, especially among the mammalian species. 4. Immunoassay procedures have failed to show any species cross-reactivity when tested against antiserum prepared in response to either the bovine or chick intestinal calcium-binding protein.     

The pig yolk sac II

Summary Since previous morphological studies have revealed abundant rough endoplasmic reticulum in the yolk sac endoderm, pig yolk sac explants from 30 day old embryos were incubated for 3–12 h with [³H]-l-leucine in order to study their protein biosynthesis. They were fractionated into a 12,000×g-pellet, 105,000×g-pellet, and supernatant. Newly synthesized proteins in these tissue fractions, and proteins discharged into the culture medium, were analysed with the aid of scintillation technique and identified by column chromatography, SDS-polyacrylamide gel electrophoresis with urea, isoelectrofocusing, and 2D-electrophoresis. Most of the radioactivity incorporated into the tissue fractions was regained from the coarse pellet and was located in the molecular weight region between 70,000 and 45,000 daltons, indicating that most of the newly synthesized proteins are membrane bound and include albumin. Albumin, an acid protein of a MW around 80,000 daltons, and many neutral and basic peptides were present in the culture medium. The yolk sac endodermal cells of the pig synthesize less proteins than those of the cat, although the pig cells display much larger amounts of endoplasmic reticulum.     

Isolation and characterization of pig enamelins.

Enamel proteins were extracted from pig developing enamel by sequential extraction procedures. Two proteins identified as enamelins by slab-gel electrophoresis (Mr 67,000 and 63,000) were separated from amelogenins by gel sieving and ion-exchange chromatography. Their enamelin characteristic was confirmed by hydroxyapatite-binding studies and amino acid analysis. Degradation of extracted enamel proteins was also studied in vitro. The larger of the two enamelins appeared to be resistant to degradation by endogenous enamel proteinases. Hydroxyapatite showed strong binding with the enamelins, but did not prevent the degradation of the Mr-63,000 enamelin. These results indicate that at least one high-Mr enamelin in pig developing enamel is a source of enamelin breakdown products.     

Purification and structure of caltrin-like proteins from seminal vesicle of the guinea pig

Two different small proteins that cross-react with the antiserum against bovine caltrin (calcium transport inhibitor) have been purified from the seminal vesicle contents of the guinea pig. The primary structure and some molecular characteristics of the pure proteins are reported. The two proteins interact with concanavalin A indicating the presence of carbohydrates in their molecules. Chemical deglycosylation with trifluoromethanesulfonic acid, after reduction and carboxymethylation, results in complete loss of affinity for the lectin. Removal of sugar components from the structure destroys the ability of caltrin-like proteins to react with antibodies to bovine caltrin. The protein moving faster on polyacrylamide gel electrophoresis is designated guinea pig caltrin I, the other is II. They contain 45 and 55 amino acids, and the molecular weights of the peptide portions are 5082 and 6255, respectively. Although they have entirely different amino acid sequences, they share some common features: recognition by rabbit antibodies to bovine caltrin, the predominance of basic residues and the presence of 3 cysteine residues in fraction I and 8 in fraction II. The proteins have pI values of 9.5 and 10.2, respectively, which are consistent with the amino acid composition. The two pure fractions are approximately equally effective, on a weight basis, as inhibitors of 45Ca2+ uptake by guinea pig spermatozoa. The data presented reinforce the hypothesis that caltrin-like proteins are responsible for the previously reported (Coronel, C.E., San Agustin, J., and Lardy, H.A. (1988) Biol. Reprod. 38, 713-722), calcium-transport inhibitor activity detected in reproductive tract fluid from adult male guinea pigs.     

Guinea pig intestinal iron binding protein

An iron binding protein, isolated from guinea pig intestinal mucosa, was compared to guinea pig transferrin. Both had a molecular weight of approximately 80,000. The intestinal iron-binding protein consisted of 2 subunits of equal molecular weight; transferrin had no subunits. Transferrin showed an absorbance peak at 470 nm; the intestinal iron-binding protein had no visible absorbance but did have a peak at 336 nm. Electron spin resonance spectra of the two proteins dfffered. Significant differences on amino acid analysis were also identified.     

Comparative studies of pig platelet membrane proteins

The proteins and glycoproteins of pig platelet membranes have been studied using gel electrophoretic techniques. A nomenclature is suggested from the apparent molecular weights estimated by one-dimensional electrophoresis. Isoelectric focusing showed that the majority of the proteins are in the 4.0-7.0 pH range. Subunits have been inferred from oligoproteins by two-dimensional, reduced-nonreduced, electrophoresis techniques. High resolution two dimensional electrophoresis combining isoelectric focusing and sodium dodecyl sulphate allows the observations of 60 polypeptide bands. An identification of some of those bands based on a correlation from reported human blood platelet membrane proteins is presented for comparison.     

Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig.

1. Soluble proteins showing binding activity to 2-isobutyl-3-methoxypyrazine have been purified to homogeneity from rabbit and pig nasal tissue; their characteristics are similar to the bovine odorant-binding protein and are to be considered members of the same family. 2. The rabbit protein is a homodimer with subunits of Mr 19k and an isoelectric point of 4.7, whereas the pig protein appears to consist of a single polypeptide chain of Mr 22k and an isoelectric point of 4.2. 3. Both proteins bind 2-isobutyl-3-methoxypyrazine with dissociation constants in the micromolar range. 4. Antibodies against the bovine OBP react well with the rabbit protein, and slightly with the porcine one.     

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

Comparative proteomic analysis of kidney development-related proteins in the pig

The development of the kidney is a complex process that serves as a model organ system for understanding many basic developmental mechanisms, and the pig kidney provides a useful and relevant model of kidney development and function. However, the molecular cascades involved in kidney development during embryonic development in the pig have not been elucidated fully. To better understand the molecular events associated with kidney development, we evaluated changes in gene expression during kidney development (days E40, E70, and E93) and compared these expressions with adults using two-dimensional gel electrophoresis. The functionally regulated proteins were identified by comparing differentially expressed proteins in embryonic kidneys vs. adult kidney. In addition, a representative set of the proteins was subjected to liquid chromatography tandem mass spectrometry analysis. Furthermore, the identified proteins were categorized according to their biological processes and molecular functions. Interestingly, 10 of the 25 proteins identified were apoptosis and actin cytoskeleton-related proteins, such as GRP75, α-fetoprotein, ANXA2, ANXA4, DDAH2, DJ-1, SOD2, cofilin1, vil1, and calbindin1. Based on these results, the proteomic approach was applied to identify specific protein expression changes in kidney tissues during development, and the expressional changes of these embryonic kidney proteins were found to be closely associated with the regulation of kidney development.     

Differences between antigenic determinants of pig and cat zona pellucida proteins

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.     

Phosphorylation of cytoplasmic ribonucleoproteins in guinea pig adrenal cells. Effect of ACTH

Phosphorylation of proteins bound with cytoplasmic ribonucleoproteins (RNP) of guinea pig adrenal cells activated by ACTH was studied. Incorporation of 32P into ribosomes and mRNP (informosomes) in check samples is not high. Phosphorylation of ribosomes, informosomes and free proteins which have separated from ribonucleoproteins sharply grow after corticotropin addition. Specificity of translation in the activated adrenal cells is supposed to be related to mRNP phosphorylation.     

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.     

Evidence of the existence of a high-density lipoprotein transformation factor in pig and rabbit plasma

Experiments have been conducted to elucidate the incubation conditions necessary for the transformation of pig high-density lipoproteins (HDL) from particles resembling the HDL3 subfraction of human plasma into particles resembling human HDL2. All incubations were performed at 37 degrees C for 24 h in the presence of 0.002 M p-chloromercuriphenylsulphonic acid, a chemical inhibitor of lecithin-cholesterol acyltransferase. There was no transformation when pig HDL (1.09 less than d less than 1.21 g/ml) was incubated either in isolation or in the presence of bovine serum albumin. In addition, there was no transformation when the 1.25 g/ml infranatant of pig plasma was added to the incubation. In the presence of the 1.25 g/ml infranatant of rabbit plasma, by contrast, a significant proportion of the pig HDL was transformed into HDL2. The possibility that this result may have been the consequence of the lipid-transfer activity in rabbit plasma was excluded by the observation that a partially purified preparation of rabbit lipid-transfer protein was ineffective in promoting the HDL transformation. Experiments were also performed with the 1.09 g/ml infranatant of pig plasma. This fraction contained HDL and the plasma proteins but no other lipoprotein. In contrast to the absence of change in incubations of a mixture containing isolated pig HDL (1.09 less than d less than 1.21 g/ml) and the 1.25 g/ml infranatant of pig plasma, during incubation of the 1.09 g/ml infranatant there was an obvious formation of HDL2. It has been concluded that there exists in plasma an HDL-transformation factor which is distinct from both lecithin-cholesterol acyltransferase and the lipid-transfer protein. This factor was detectable in the 1.25 g/ml infranatant of rabbit plasma and in the 1.09 g/ml infranatant of pig plasma. As the factor was not detected in isolated pig HDL or in the 1.25 g/ml infranatant of pig plasma, the 1.21-1.25 g/ml fraction of plasma may be a useful starting point for further investigation of this factor.     

Localization of chorionic pregnancy-associated glycoprotein family in the pig

The objective of this study was to localize the immuno-positive porcine PAG (pPAG) proteins within chorionic cells throughout the intensive placenta development as pregnancy advances (16-61 days post coitum - dpc). Placental sections were used for double fluorescent histochemistry with selected primary rabbit anti-pPAG sera. The polyclonals were created against recombinant pPAG2 antigen or various secretory porcine native chorionic antigens produced in vitro. Among placental cells stained with fluorescent propidium iodine, the positive pPAG immuno-complexes were visualized by Alexa 488 fluorochrom - conjugated to secondary anti-rabbit goat immunoglobulins. This is the first report concerning cellular localization of the pPAG protein family within diffuse epitheliochorial placenta development throughout the first half of pregnancy in the pig. Fluorescent immuno-positive pPAG signals have been restricted to chorionic cell layers (branched mushroom-like and finger-like structures) that generate a epitheliochorial feto-maternal surface augmented by maternal endometrium interdigitations with the gestation progress in the pig. These results suggest that the pPAG proteins robustly expressed in chorionic cells are involved in the regulation of intensive development of diffuse porcine placenta during the first half of pregnancy.     

Protein patterns of pig oocytes during in vitro maturation

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.