Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain

We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.     

Fatty-acid-binding protein from bovine brain. Amino acid sequence and some properties

A fatty-acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified FABP behaved as an anionic protein with an apparent molecular mass of 14.7 kDa; its complete amino acid sequence was determined and microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP and bovine mammary-derived growth inhibitor (MDGI).     

Purification and identification of two pertussis-toxin-sensitive GTP-binding proteins of bovine spleen

Two alpha subunits of GTP-binding proteins were purified from bovine spleen membranes. Both proteins were ADP-ribosylated by pertussis toxin in the presence of beta gamma subunits. The major protein had a molecular mass of 40 kDa and its immunological reactivity and fragmentation pattern by limited proteolysis were identical with those of the alpha subunit of Gi2. The minor protein had a molecular mass of 41 kDa and its partial amino acid sequences completely matched with those predicted from human and rat Gi3 alpha cDNAs.     

Microfibril-associated protein 4 is present in lung washings and binds to the collagen region of lung surfactant protein D.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Lung surfactant protein D (SP-D) was also purified from lung washings, and calcium-dependent binding was demonstrated between bMFAP4 and SP-D. hMFAP4 was cloned, and recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. No binding was seen to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D, indicating that the interaction between MFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. Our findings indicate that MFAP4 has two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.     

A brain protein (P30) that immunoreacts with a polyclonal anti-pancreatic carboxypeptidase A antibody shows properties that are shared with tubulin carboxypeptidase

A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 ± 0.2). When the tubulin carboxypeptidase preparation was subjected to gel filtration chromatography under low salt concentration, P30 behaved as a protein of molecular mass 38 kDa whereas tubulin carboxypeptidase eluted at a position of 75 kDa molecular mass. However, when the chromatography was performed at relatively high salt concentration they behaved as proteins of 49 and 56 kDa, respectively. We considered that P30 may be an inactive monomeric form of the dimeric tubulin carboxypeptidase. However we can not rule out the possibility that it represents another carboxypeptidase not yet described.     

Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60°C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.     

Identification of the neuronal protein with antigenic properties of latrotoxin

Monospecific antibodies against neurotoxin from the black widow spider venom (alpha-latrotoxin) have been used to identify a neuronal protein (s) with latrotoxin-like epitopes. As determined by ELISA, the protein is localized in cytoplasmic fraction of bovine brain. It was partially purified by anion-exchange chromatography and preparative SDS-electrophoresis. Immunoblotting data indicate that this neuronal acidic protein has molecular mass of ca. 100 kDa.     

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.     

Identification and partial purification of a band 3-like protein from rabbit renal brush border membranes

A monoclonal antibody against the membrane domain of human erythrocyte band 3 was tested for its ability to bind to rabbit renal brush border membranes. A single brush border protein with a molecular mass of 43 kDa was recognized by the band 3 antibody. Using DNase I coupled to an agarose-bead support this 43-kDa protein was partially purified by removing actin and a number of actin-bound proteins from the brush border membranes. The partially purified 43 kDa-band was eluted from sodium dodecyl sulfate-polyacrylamide gels and used to make a highly sensitive and specific guinea pig antiserum. This antiserum, but not serum from control guinea pigs, cross-reacts with purified band 3 from human, rabbit, and bovine erythrocytes confirming the immunologic similarity among these proteins. The 43-kDa protein can be stained by the periodic acid-Schiff base method and binds wheat germ agglutinin and concanavalin A, demonstrating that it is a glycoprotein. Furthermore, in the absence of dithiothreitol, the immunoreactive brush border protein migrates with a molecular mass of 86 kDa on an sodium dodecyl sulfate-polyacrylamide gel suggesting that under nonreducing conditions it exists as a dimer. The 43-kDa protein could be solubilized in octyl glucoside and was further purified using gel filtration chromatography. The amino acid composition of the 43-kDa brush border protein was obtained, and its similarity with erythrocyte band 3 is discussed.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

A bovine plasminogen activator of atypical molecular mass ( approximately 45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072-1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.     

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

Truncated mutants of the colicin A lysis protein are acylated and processed when overproduced

Abstract Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca²⁺-dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca²⁺ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis.     

Purification and properties of mouse liver coproporphyrinogen oxidase

Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.     

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.     

Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2

Presence or absence of three distinct bovine seminal heparin-binding proteins (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility-associated antigenin bovine seminal fluid as a unique DNase I-like protein. We now report purification and identification of a 24 kDa seminal heparin-binding protein (HBP-24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identity to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding proteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm membranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin-binding protein increased under nonreducing SDS-PAGE to approximately 21 kDa, characteristic of the reported molecular mass of TIMP-2. To our knowledge, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate previous reports regarding the site of production of heparin-binding proteins that are related to bull fertility, and suggest that TIMP-2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition.     

Purification and Characterization of a Novel Brain-Specific 14-kDa Protein

A new acidic protein specifically present in the brain was purified to homogeneity from bovine brain. The apparent molecular mass was estimated to be 14 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 57 kDa by gel filtration, a finding suggesting that it exists as a tetramer under physiological conditions. The protein had a high content of Glu and Pro, and its pI was 4.3. The first six amino acid residues of the protein were Met-Asp-Val-Phe-Met-Lys, and the amino terminal was blocked. The distribution of the protein examined by Ouchterlony gel immunodiffusion indicates that it is present specifically in brain, including rat, human, and bovine, but could not be detected in 10 other rat tissues examined. The protein was absent in Purkinje cell bodies, as examined by electron microscopic immunocytochemistry, but was present in nerve terminals that make synapse-like contacts with Purkinje cells and in neurons with dark granules in the globus pallidus of the rat.