The purification of calcium-binding protein from the uterus of the laying hen.

Calcium-binding proteins from the chick intestine and uterus of the laying hen (Gallus domesticus) were isolated and purified by identical methods. Proteins from each source were shown to exhibit identical electrophoretic mobility and were immunologically identical. Estimation of molecular size by gel filtration indicated a value of approximately 28,000 daltons for both the chick intestinal and hen uterine calcium-binding proteins. The amino acid compositions of chick intestinal and hen uterine calcium-binding proteins were essentially identical.     

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.     

Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins.

Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.     

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.     

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.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

Isolation and purification of calcium-binding protein from electroplax of Electrophorus electricus.

An acidic calcium-binding phosphoprotein has been isolated from a cholinergic tissue, electroplax from Electrophorus electricus. The purification procedures included (NH4)2SO4 fractionation, boiling treatment, ECTEOLA-cellulose chromatography, and gel filtration on Sephadex G-100. Experiments were performed to compare this protein and a calcium-binding protein isolated from mammalian brain, adrenal medulla, and testis. These experiments showed that the two proteins were identical in terms of molecular weight (14 000), calcium-binding dissociation constant (kd=2.1-10(-5) M), electrophoretic mobility at pH 8.7 in 15% polyacrylamide gels, and phosphorus content (1 mol phosphorus per mol protein). In addition, the two proteins had similar amino acid compositions and peptide maps. Although the electroplax protein was not present in eel skeletal muscle, preliminary experiments indicated that small amounts of the protein were present in other eel tissues, namely brain, liver and spleen. These results suggest an identity between the electroplax and mammalian calcium-binding proteins and extend the findind of comparatively large amounts of the protein from mammalian nervous tissue to a cholinergic nervous tissue, electroplax. The close similarity of the proteins suggests a conservation of structure during evolution which may be required to fulfill a role in neuronal function.     

The response of the small intestine to vitamin D. Isolation and properties of chick intestinal polyribosomes

Undegraded polyribosome preparations may be obtained from chick intestinal mucosa if ribonuclease activity is strictly controlled. This is best achieved by homogenization of the mucosa directly in rat liver cell-sap. 2. The extent of amino acid incorporation by chick intestinal polyribosomes is greatly influenced by the source of the cell-sap. Sephadex-treated intestinal cell-sap caused impaired incorporation and release of completed polypeptide chains, whereas Sephadex-treated rat liver cell-sap promoted the polymerization of up to 90 amino acids per ribosome. Under optimum conditions 30-35% of the nascent polypeptide chains are completed and released. 3. The preparation of an antiserum against the calcium-binding protein formed in response to vitamin D is described. It is shown that the antiserum is highly specific for calcium-binding protein. 4. This antiserum was used to investigate the ability of chick intestinal polyribosomes to synthesize calciumbinding protein. Only polyribosomes from chicks receiving vitamin D have the ability to synthesize calcium-binding protein. Moreover, the product formed in vitro has the same electrophoretic mobility as calcium-binding protein synthesized in vivo. 5. It is concluded that one of the main functions of vitamin D in the small intestine is to induce the synthesis de novo of calcium-binding protein.     

Studies on the synthesis and degradation of a high molecular weight, histidine-rich phosphoprotein from mammalian epidermis

The synthesis and subsequent fate of the histidine-rich proteins, which form a major component of keratohyalin granules in mammalian epidermis, have been studied in the guinea-pig and new-born rat. In both species the protein first synthesised is of very high molecular weight, approximately 340 000. It is short-lived and breaks down to lower molecular weight proteins 1-2 days after its synthesis. These smaller proteins differ in the two species. In the guinea-pig, the high molecular weight protein breaks down to proteins of molecular weight 250 000 and 200 000, which are themselves unstable and break down to low molecular weight species, probably amino acids. The initial breakdown of the high molecular weight protein coincides with the dispersion of the keratohyalin granules and the transition of the granular cell into the stratum corneum. This high molecular weight histidine-rich protein has been purified to homogeneity, despite its instability to several treatments during purification. The protein is highly phosphorylated, containing 6 mol% of phosphoserine, but is otherwise very basic. The possibility that dephosphorylation of the protein produces highly basic matrix proteins in the stratum corneum is discussed.     

Purification of mammalian filamin. Similarity to high molecular weight actin-binding protein in macrophages, platelets, fibroblasts, and other tissues

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.     

Preparation and characterization of calcium-binding and other hydrophobic proteins from synaptic membranes

A number of hydrophobic proteins have been separated and purified to varying degrees from synaptic membranes derived from bovine brain. The proteins, which have been obtained using preparative acrylamide gel electrophoresis, have been analyzed for molecular weight, amino acid composition, peptide mapping, N-terminal amino acids, and for their ability to bind calcium and ATP. A number of the proteins bound calcium, the greatest binding being associated with a component having a molecular weight of 1.5 · 10⁴, a binding capacity of 4 calcium/molecule, and a K_m of 1.5 · 10^?5 M. An acidic tryptic peptide derived from this protein was evidently responsible for the calcium-binding. ATP binding appeared to be confined largely to the higher molecular weight proteins. From the peptide mapping there appears to be a similar acidic component in a number of the proteins exhibiting calcium-binding. ATP-binding was associated mainly with the high molecular weight proteins, particularly those which consisted of numerous basic tryptic peptides.     

Isolation, characterization, and developmental expression of pig intestinal fatty acid-binding proteins

The goal of this study was to characterize and quantify intestinal fatty acid-binding proteins of the pig. Small intestinal mucosa from 13-19 kg pigs was homogenized and centrifuged to obtain cytosol. Isolation of fatty acid-binding proteins from delipidated cytosol was achieved using molecular sieve, oleic acid affinity, and ion exchange chromatography. Fatty acid-binding protein isolation was monitored using a fatty-acid binding assay in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antisera to rat liver-fatty acid-binding protein cross reacted with an isolated intestinal fatty acid-binding protein of Mr = 13,000, whereas antisera to rat intestine-fatty acid-binding protein was not cross reactive with isolated pig intestinal proteins. These experiments identify a pig intestinal fatty acid-binding protein that exhibits strong immunochemical similarity to rat liver-fatty acid-binding protein. Cytosol prepared from intestinal mucosa of pigs at -4, 2, 4, 7, 15, 22, 28, and 35 d of age was assayed for fatty acid-binding protein activity. Preweaning fatty acid-binding protein activity in cytosol was maximal at 7 days of age when expressed as total jejunal fatty acid binding per kilogram bodyweight, intestinal or mucosal weight or milligram total protein. After weaning (21 d), fatty acid-binding protein activities declined to 28 days, but increased again by 35 days. Total soluble fatty acid-binding protein activity in pig intestine is regulated during postnatal development and this may account in part for the altered intestinal absorption of lipids observed in young pigs at weaning.     

Structure of the human brain calcium-binding protein calretinin and its expression in bacteria

Calbindin D28k and calretinin are two closely related intracellular calcium-binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin-D-dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31,520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium-binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 x 10(-9) amino acid-1 year-1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium-binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.     

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.     

The flavin-containing monooxygenase expressed in pig liver: primary sequence, distribution, and evidence for a single gene

The primary sequence of the flavin-containing monooxygenase expressed in pig liver has been derived from the nucleotide sequence of cloned cDNA. The derived sequence is composed of 532 amino acids and represents a protein having a molecular weight of 58,952. The complete sequence was obtained from a single clone containing 2070 bases. A second clone, obtained from an independent library, yielded an identical sequence for the 1374 bases present. The amino acid composition compiled from the derived sequence is very similar to that obtained previously from the purified protein. In addition, a 10 amino acid sequence in a peptide formed from the purified protein by digestion with V8 protease exactly matches the derived sequence for residues 309-318. The flavin-containing monooxygenase expressed in pig liver is also expressed in pig lung and kidney as determined by analysis of both microsomal proteins and mRNA. The ratio of mRNA to protein for the enzyme in kidney is about 5 times greater than the same ratio for liver and about twice the ratio for lung. The reasons for these differences are not understood. Southern analysis of genomic DNA indicates that there is a single gene encoding the flavin-containing monooxygenase expressed in pig liver. Therefore, the broad activity of this enzyme in liver appears to be the result of the catalytic diversity of a single protein.     

The PO Protein of Chick Sciatic Nerve Myelin: Purification and Partial Characterization

Abstract: The PO protein of the myelin of chick sciatic nerve was isolated and purified by propanoic acid extraction of peripheral nervous system (PNS) myelin, delipidation, Sepharose CL-6B chromatography in the presence of sodium dodecyl sulfate (SDS), and preparative SDS-polyacrylamide gel electro-phoresis (PAGE). Approximately 15% of the PO protein in the sciatic nerve myelin was recovered in a homogeneous state. The purified protein monomer has an apparent molecular weight of 32.1K as determined by gel electrophoresis. The PO protein undergoes extensive aggregation during exhaustive dialysis and freeze-drying and yields stable dimers, trimers, and tetramers. The aggregation of the PO protein after freeze-drying is independent of the presence of a reducing agent (2-mercaptoethanol) in the solubilizing medium. The PO protein is a glycoprotein. The amino acid composition of the chick PO protein indicates a definite species difference when compared with mammalian PO proteins although the NH₂-terminal isoleucine residue seems to have been retained during evolution.     

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3 modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.     

A tissue culture system for studies of the regulation of ether-linked and ester-linked aliphatic moieties in glycerolipids

A protein has been detected in chick brain which is immunologically identical to the vitamin D-induced calcium-binding protein found in intestinal tissue. It is present in highest concentration in the cerebellum and at lower levels in the remainder of the brain. Brain calcium-binding protein has the same molecular weight and electrophoretic properties as intestinal calcium-binding protein. Although the synthesis of intestinal calcium-binding protein is totally dependent upon a source of vitamin D, this has not yet been shown for brain calcium-binding protein. The total calcium-binding protein content of the cerebellum of chicks fed a vitamin D-free diet continued to increase during growth from 1 to 5 weeks, and is not responsive to exogenous vitamin D or 1,25-dihydroxyvitamin D.     

Sequence and evolution of guinea pig preproinsulin DNA

Guinea pig insulin exhibits an unusually high degree of divergence from the conserved insulins of other mammals. cDNA clones encoding guinea pig preproinsulin were isolated, and their nucleic acid sequences were determined. Comparisons of the nucleic acid sequence and its predicted amino acid sequence with sequences encoding insulins of other species revealed that the gene encoding guinea pig preproinsulin evolved from the same ancestral mammalian gene as other known mammalian insulin genes.