Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C.

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.     

Two low-molecular-weight Ca2+-binding proteins isolated from squid optic lobe by phenothiazine--Sepharose affinity chromatography.

We have isolated two Ca2+-binding proteins from squid optic lobes, each of which is also able to bind phenothiazines in a Ca2+-dependent manner. These proteins have each been purified and partly characterized. One of the proteins corresponds to calmodulin, in that it has a similar amino acid content to bovine brain calmodulin, including a single residue of trimethyl-lysine, it co-migrates with bovine calmodulin both on alkaline-urea- and on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis, and will activate calmodulin-dependent phosphodiesterase. The second protein has the same subunit molecular weight as calmodulin, as determined by SDS/polyacrylamide-gel electrophoresis, Mr 17 000, but migrates more slowly than this protein on alkaline-urea-gel electrophoresis. It has an amino acid composition distinct from calmodulin, containing no trimethyl-lysine, its CNBr fragments migrate on alkaline gels in a pattern distinct from those of calmodulin and it shows little ability to activate phosphodiesterase. The u.v.-absorption spectra of the proteins indicate the absence of tryptophan and the presence of a high phenylalanine/tyrosine ratio in each. Both proteins also bind 3-4 calcium ions/mol at 0.1 mM-free Ca2+ and each binds chlorpromazine in a Ca2+-dependent manner.     

67 kDa calcimedin, a new Ca2+-binding protein.

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.     

Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Branched-chain oxo acid dehydrogenase was purified from Pseudomonas aeruginosa strain PAO with the objective of resolving the complex into its subunits. The purified complex consisted of four proteins, of Mr 36,000, 42,000, 49,000 and 50,000. The complex was resolved by heat treatment into the 49,000 and 50,000-Mr proteins, which were separated by chromatography on DEAE-Sepharose. The 49,000-Mr protein was identified as the E2 subunit by its ability to catalyse transacylation with a variety of substrates, with dihydrolipoamide as the acceptor. P. aeruginosa, like P. putida, produces two lipoamide dehydrogenases. One, the 50,000-Mr protein, was identified as the specific E3 subunit of branched-chain oxo acid dehydrogenase and had many properties in common with the lipoamide dehydrogenase LPD-val of P. putida. The second lipoamide dehydrogenase had Mr 54,000 and corresponded to the lipoamide dehydrogenase LPD-glc of P. putida. Fragments of C-terminal CNBr peptides of LPD-val from P. putida and P. aeruginosa corresponded closely, with only two amino acid differences over 31 amino acids. A corresponding fragment at the C-terminal end of lipoamide dehydrogenase from Escherichia coli also showed extensive homology. All three peptides had a common segment of eight amino acids, with the sequence TIHAHPTL. This homology was not evident in any other flavoproteins in the Dayhoff data base which suggests that this sequence might be characteristic of lipoamide dehydrogenase.     

The purification and complete amino acid sequence of the 9000-Mr Ca2+-binding protein from rat placenta. Identity with the vitamin D-dependent intestinal Ca2+-binding protein.

A 9000-Mr Ca2+-binding protein was isolated from rat placenta and purified to homogeneity by h.p.l.c. procedures. The complete amino acid sequence was established for the 78-residue placental protein. A sequence analysis of a minor component of the rat intestinal Ca2+-binding protein (residues 4-78) and a tryptic peptide (residues 55-74), both purified by h.p.l.c., showed both proteins to be identical. Thus this placental 9000-Mr Ca2+-binding protein is the same gene product as the intestinal Ca2+-binding protein whose synthesis is dependent on vitamin D.     

Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin.

The interaction between type 1 plasminogen activator inhibitor (PAI-1) and fragments of vitronectin (Vn) was investigated. The PAI-1-binding domain was not destroyed when Vn was cleaved by treatment with either acid or CNBr. Acid-cleaved Vn was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by PAI-1 ligand binding. The smallest fragment (Mr 40,000) that retained PAI-1 binding function was sequenced and shown to contain the NH2 terminus of the molecule. Further cleavage of this fragment by treatment with CNBr generated a Mr 35,000 fragment (Pro52-Asp239) that did not interact with PAI-1, and a Mr 6,000 NH2-terminal fragment (Asp1-Met51) that spanned the somatomedin B domain and contained the RGD (cell binding) sequence. The purified Mr 6,000 fragment competed with immobilized Vn for PAI-1 binding, and formed complexes with activated PAI-1. These complexes could be immunoprecipitated by antibodies to PAI-1. Synthetic peptides containing the RGD sequence had no effect on the binding of this fragment to PAI-1. These results suggest that the cell-binding and PAI-1 binding sequences of Vn occupy distinct regions in the NH2-terminal somatomedin B domain of the molecule.     

Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.     

Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane.

A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.     

Isolation and characterization of native adult osteonectin

Noncollagenous bovine bone proteins were obtained from EDTA-solubilized extracts of adult bovine bone in the absence of denaturants. Native osteonectin was isolated from the noncollagenous bone proteins by ion-exchange chromatography using DEAE-Sephadex A-50 and DEAE-Sephadex A-25, followed by gel filtration on Sephadex G-100. Comparison of the physical and chemical properties (i.e. electrophoric mobility, amino acid composition and pI) of this protein with those reported by Termine et al. (Termine, J.D., Belcourt, A.B., Conn, K.M., and Kleinman, H.K. (1981) J. Biol. Chem. 256, 10403-10408) indicate that this protein is osteonectin. Sedimentation equilibrium analyses in the presence of 6 M guandinium chloride, 10 mM Ca2+, 10 mM EDTA, or 0.15 M NaCl all yielded a molecular weight of 29,100 +/- 900. 125I-Osteonectin underwent saturable and exchangeable binding to hydroxyapatite and calf skin collagen. Eleven mg of 125I-osteonectin bound to 1.0 g of hydroxyapatite with a Kd of 8 X 10(-8) M. The intrinsic fluorescence of bovine osteonectin was partially quenched when micromolar Ca2+ was added, indicating a high affinity Ca2+ interaction. Native osteonectin was found to reduce the rate of hydroxyapatite crystal seeded growth by 50% (1 IU) at a concentration of 1.6 X 10(-7) M at pH 7.4, 37 degrees C in 0.15 M NaCl. This makes osteonectin one of the most potent inhibitors of hydroxyapatite formation presently known and more than 5 times as effective as bone Gla protein (1 IU = 8 X 10(-7) M).     

Purification and characterization of a novel Ca2+-binding protein (CBP-18) from bovine brain

A novel Ca2+-binding protein (CBP-18) has been identified and purified from bovine brain. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein consists of a single band of apparent Mr 18,000 in the presence of Ca2+ or 20,000 in the presence of EGTA. CBP-18 contains one high affinity Ca2+-binding site, measured at 10(-5) M Ca2+ in the presence of 1 mM Mg2+ and 0.1 M K+. The amino acid composition and UV absorption spectrum distinguish CBP-18 from other Ca2+-binding proteins identified in brain. The protein has an extinction coefficient epsilon 1% 279 nm = 4.9 and contains 1 tryptophan/mol, 5 tyrosines/mol, and no trimethyllysine. CBP-18 does not interact with or activate calmodulin-stimulated phosphodiesterase. However, available evidence suggests that CBP-18 binds to other component(s) present in the brain extract in a Ca2+-dependent manner.     

Localization of viral-envelope-glycoprotein-binding sites in fibronectin.

Purified viral-envelope glycoproteins from influenza A virus were found to bind to two fragments of the fibronectin molecule. Human plasma fibronectin was digested by leucocyte cathepsin G, and three different fragments, of Mr 30000, 40000 and 12000-140000, with specific binding functions were isolated. Micelles of radiolabelled influenza A glycoprotein were allowed to bind to these fragments immobilized on polystyrene micro-titre wells. The C-terminal 120000-14000-Mr fragments that carry the cell-binding activity bound viral proteins most efficiently, whereas the 40000-Mr gelatin-binding fragment bound considerably less. The N-terminal 30000-Mr Staphylococcus aureus-binding fragment was negative in the assays. Laminin, a basement-membrane protein, also bound viral proteins, though less effectively than fibronectin. The binding was abolished if laminin or fibronectin fragments were pretreated with neuraminidase. This suggests that the sialic acids in the sugar moieties of these glycoproteins are involved in the binding. The affinity of viral-envelope glycoproteins for certain domains of fibronectin and for laminin may play a role in virus-cell interactions.     

Isolation of mammalian calelectrins: a new class of ubiquitous Ca2+-regulated proteins

In a new approach to isolating proteins which participate in the Ca2+-dependent regulation of membrane traffic in animal cells, two new Ca2+-binding proteins (Mr 67 000 and 32 500) have been identified in and purified from bovine liver, brain, and adrenal medulla. These proteins specifically and reversibly bind to chromaffin granule membranes at low Ca2+ concentrations (half-maximal binding at 5.5 microM Ca2+) and greatly potentiate the Ca2+-induced aggregation of these membranes at higher concentrations (above 10 microM). In the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, the isolated proteins have Stokes radii of 3.40 nm (Mr 67 000) and 2.53 nm (Mr 32 500) as estimated by gel filtration and therefore occur as monomers. They are slightly acidic proteins with pI's of 5.85 and 5.60. In bovine tissues, both proteins and a third protein of Mr 35 000 cross-react immunologically with each other and with Torpedo calelectrin (Mr 34 000) and are therefore identified as mammalian calelectrins. In all tissues of Torpedo marmorata tested, only a single molecular mass form of calelectrin exists, whereas multiple forms of calelectrin exist in mammalian tissues, indicating gene duplication during evolution. We suggest that the evolutionary conservation and diversification, the high tissue concentrations, and the Ca2+-specific interactions of the calelectrins make them candidates for Ca2+-dependent regulators of membrane events in animal cells.     

Structural characteristics and intermolecular organization of human pulmonary-surfactant-associated proteins.

The structural relationships and intermolecular organization among the proteins associated with pulmonary surfactant are largely unknown. We studied the pulmonary-surfactant-associated proteins in the bronchoalveolar lavage fluid obtained from a patient with the clinical syndrome of alveolar proteinosis. The major proteins with Mr values of 32,000-36,000 and 62,000 formed thiol-dependent complexes (Mr greater than 400,000) with intermolecular disulphide bonds present in the collgenase-sensitive domains of these proteins. In contrast, other proteins, which were collagenase-insensitive, formed thiol-dependent oligomers that were not covalently linked to the major proteins. The associations of these proteins in the surfactant of a normal individual were similar. By amino acid analysis, two-dimensional peptide mapping and bacterial-collagenase digestion the 32,000-36,000-Mr and 62,000-Mr proteins were nearly identical. Differences in CNBr cleavage products suggested that the larger of the proteins was formed by non-disulphide, covalent, cross-links in the collagenase-sensitive domains of the 32,000-36,000-Mr proteins. Thus the evidence suggested that the lipid-associated proteins of Mr 32,000-36, 000 contained both disulphide and non-disulphide cross-links in the collagen-like N-terminal region of the proteins and form higher-Mr complexes. This organization may support the three-dimensional conformation of surfactant in the alveolar space.     

Structure of alpha-polymer from in vitro and in vivo highly cross-linked human fibrin.

Peptides derived from plasmic and cyanogen bromide (CNBr) cleavage of highly cross-linked fibrin were isolated and characterized by sodium dodecyl sulfate-gel electrophoresis, amino acid analyses, cyanoethylation, and NH2-terminal analyses. Extended plasmic digestions of human fibrin containing four epsilon-(gamma-glutamyl)lysine cross-links per molecule produced a peptide of alpha-chain origin (Mr congruent to 21,000) which was comprised of a small donor peptide cross-linked to the acceptor site peptide from the middle of the alpha-chain. CNBr cleavage of highly cross-linked in vitro fibrin or of fibrin from a spontaneously formed in vivo arterial embolus produced about three cross-linked species of molecular weights 30,000 to 40,000, each of which contained the largest CNBr fragment (Mr = 29,000) from the alpha-chain. The predominant cross-link-containing CNBr fragments derived their donor group from the near COOH-terminal region of the alpha-chain as judged by difference amino acid compositions and NH2-terminal analyses. Additionally, cross-linked fragments of molecular weights 68,000 to 70,000 which appeared to contain two acceptor site peptides (Mr = 29,000) were detected in minor amounts in the CNBr digests of fibrin formed from whole plasma or from purified, plasminogen-free fibrinogen. No larger polymeric cross-linked CNBr fragment was generated from any of the highly cross-linked fibrin preparations examined. A model for the predominant mode of alpha-chain polymerization is proposed.     

Identification of pre-osteonectin produced by cell-free translation of fetal porcine calvarial mRNA

The cell-free biosynthesis of the bone protein osteonectin was studied using mRNA from fetal porcine calvariae. Total RNA was extracted from the calvariae with guanidinium thiocyanate and was partially purified by precipitation with acid/ethanol. Translations were performed using the reticulocyte lysate system and were optimized with respect to mRNA concentration and K+ (70 mM) and Mg2+ (0.6 mM) concentration. Cell-free synthesized osteonectin, radiolabeled with [35S]methionine, was specifically immunoprecipitated with rabbit antiserum to porcine osteonectin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. When analyzed under reduced conditions, the translated protein migrated with an Mr 45,000 compared to an Mr 39,000 for cell-synthesized osteonectin. When translated in the presence of microsomal membranes, the immunoprecipitated osteonectin co-migrated with the cell-synthesized osteonectin, indicating that a signal sequence of about 45-50 amino acids (Mr 6,000) had been removed. Under nonreduced conditions the pre-osteonectin co-migrated with osteonectin (Mr 39,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that a highly folded structure is retained by disulfide bridges under denaturing conditions. The relationship between the immunoprecipitated pre-osteonectin from the cell-free translations and both the cell-synthesized and tissue-extracted osteonectin was confirmed by one-dimensional peptide mapping of Staphylococcus aureus V-8 protease digestions. The results indicate that porcine osteonectin is synthesized on polysomes in a pre-osteonectin form which is translocated vectorially into microsomal vesicles and cotranslationally processed by the removal of a signal peptide.     

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.     

A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.     

Caprine acrosin. Purification, characterization and proteolysis of the porcine zona pellucida.

Acrosin purified from an acidic extract of ejaculated goat spermatozoa migrated as a single 42,000-Mr band in SDS/polyacrylamide-gel electrophoresis. Reduction and alkylation of caprine acrosin produced two polypeptides, one of Mr 40,000 (heavy chain) and the other of Mr 3700 (light chain). The light chain purified by reversed-phase h.p.l.c. was a glycosylated octadecapeptide with an amino acid sequence similar to that of the N-terminal 18 residues of porcine acrosin light chain (78% positional identity). The sequence of the N-terminal 37 amino acids of purified caprine acrosin heavy chain is similar to that of porcine acrosin heavy chain (70% positional identity through 37 residues). Studies with synthetic substrates and synthetic and natural proteinase inhibitors confirmed both the specificity of the purified proteinase for Arg-Xaa and Lys-Xaa bonds and a serine-proteinase mechanism. Purified caprine acrosin hydrolysed the 90 kDa and 65 kDa components, but did not hydrolyse the 55 kDa component of the porcine zona pellucida. The action of the enzyme on the porcine zona pellucida was indistinguishable from that previously reported for porcine acrosin.     

Isolation of two isoforms of a 21,000-dalton Ca2+-binding protein of bovine brain

Using Ca2+-dependent hydrophobic interaction chromatography we have identified a novel bovine brain Ca2+-binding protein (CaBP) composed of 21 kDa and 23 kDa polypeptides. This calciprotein was further purified by heat-treatment in the presence of Ca2+ and ion-exchange chromatography. The isolated protein exhibits a number of properties in common with proteins belonging to the calmodulin family of CaBPs, including a Ca2+-dependent electrophoretic mobility shift on SDS-polyacrylamide gel electrophoresis, retention of the ability to bind 45Ca2+ after electrophoresis and Western blotting, and a high content of acidic amino acids. We have recently isolated and characterized a 21 kDa CaBP from bovine brain and conclude that the 21 kDa and 21/23 kDa CaBPs are isoforms since they have very similar U.V. absorption spectra and amino acid compositions, and polyclonal antibodies raised in rabbits against the 21 kDa CaBP cross-react to an identical degree with the 21/23 kDa CaBP as determined by the competitive enzyme-linked immunosorbent assay (ELISA). Both proteins contain carbohydrate, but they differ in the degree of glycosylation. Tissue distribution studies indicate the presence of both 21 kDa and 23 kDa Ca2+-binding polypeptides in bovine trachea, aorta, kidney, skeletal muscle and cardiac muscle, and chicken gizzard smooth muscle.     

Purification, characterization, and the complete amino acid sequence of porcine pancreatic deoxyribonuclease

Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.