Canine thyroid fucokinase.

A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.     

Isolation and characterization of a cDNA clone for porcine thyroid peroxidase

We undertook the molecular cloning of porcine thyroid peroxidase (TPO). Four oligonucleotide probes were synthesized on the basis of amino acid sequences of 3 tryptic peptides from highly purified porcine TPO. These probes were used to screen a pig thyroid cDNA library. Seven of 16 selected clones (0.45-1.15 kb in size) reacted with all 4 probes. Nucleotide sequencing of the 1.15 kb at the 3'-end of the structural gene revealed the complementary sequence to all 4 probes as well as the nucleotides coding for the entire length of the 3 tryptic peptides. There is an open reading frame of 332 amino acid residues. On Northern blot analysis this gene codes for an mRNA species of 2.85 kb, corresponding to the anticipated size of the mRNA for the intact TPO molecule. We have therefore cloned and characterized a cDNA clone coding for approx. 36% of porcine thyroid peroxidase.     

Isolation and properties of porcine lecithin:cholesterol acyltransferase

Lecithin: cholesterol acyltransferase (LCAT, phosphatidylcholine: sterol O-acyltransferase, EC 2.3.1.43) was purified approximately 20 000-fold from pig plasma by ultracentrifugation, phenyl-Sepharose and hydroxyapatite chromatography. Purified LCAT had an apparent relative molecular mass of 69 000 +/- 2000. By isoelectrofocusing it separated into five or six bands with pI values ranging from pH 4.9 to 5.2. The amino acid composition was similar to that of the human enzyme. An antibody against pig LCAT was prepared in goat. The antibody reacted against pig LCAT and gave a reaction of partial identity with human LCAT. Incubation of pig plasma or purified enzyme with the antibody virtually inhibited LCAT activity. The same amount of antibody inactivated only 62% of the LCAT activity in human serum. Pig and human LCAT were activated to the same extent by either human or pig apolipoprotein A-I (apo-A-I) using small liposomes as substrate. Human apoA-I, however, caused a higher esterification rate for both enzymes. Using apoA-I and small liposomes as a substrate, the addition of apoC-II up to 4 micrograms/ml had no effect on the LCAT reaction, but above this concentration LCAT was inhibited. Small liposomes with phosphatidylcholine/cholesterol molar ratios of 3:1 up to 8.4:1 did not show any significant differences in the LCAT reaction, when used as substrates in the presence of various amounts of apoA-I and albumin. In contrast, the LCAT activity was significantly reduced by liposomes with phosphatidylcholine/cholesterol molar ratios below 3:1.     

Isolation and characterization of porcine beta-casein.

Porcine beta-casein was isolated by chromatography on DEAE-cellulose. The protein had a molecular weight of 24 900 as determined by gel filtration on Sephadex G-100 in guanidine-HCl. Its amino acid composition differed from bovine beta-casein especia-ly in respect to serine, alanine and leucine. In common with bovine beta-casein the N-terminal amino acid was arginine; the C-terminal was either alanine or valine, while the C-terminal of bovine beta-casein is valine. At any temperature porcine beta-casein was more sensitive to Ca2+ than bovine beta-casein, while at a fixed Ca2+ concentration porcine beta-casein aggregated at a lower temperature than bovine beta-casein. Porcine beta-casein was susceptible to hydrolysis by calf chymosin but the proteolytic specificity differed from that of calf chymosin on bovine beta-casein.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

Isolation and structure of somatostatin from porcine hypothalami.

The isolation and structure of somatostatin (GH-RIH) from pig hypothalami are described. This hormone was purified by preparative gel filtration, solvent extraction, countercurrent distribution in two solvent systems, ion-exchange and partition chromatography, and analytical gel filtration. The somatostatin activity was followed by in vitro bioassays and a radioimmunoassay. The isolated product was homogeneous chromatographically and had biological and immunological properties similar to synthetic somatostatin corresponding to the ovine hormone. The primary structure of porcine somatostatin was shown to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Other immunologically and biologically active form(s) of somatostatin were also detected.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Isolation of virulent Yersinia enterocolitica from porcine tongues.

Thirty-one tongues from apparently normal, freshly slaughtered pigs were assayed for the presence of Yersinia enterocolitica by different enrichment and postenrichment techniques. Sixteen different isolates were recovered, including six of serotype O:8, four of serotype O:6,30, two of serotype O:3 phage type IXb, and one each of serotypes O:13,7, O:18, and O:46. One isolate was not typable. Cold enrichment in phosphate-buffered saline followed by treatment with dilute KOH or subsequent enrichment in modified Rappaport broth recovered 12 and 7 isolates, respectively. Four of the same isolates were recovered from the same tongues by both procedures. Cold enrichment without a selective postenrichment treatment recovered two isolates. Direct enrichment in modified Rappaport broth or modified selenite broth was ineffective in recovering yersiniae, as no isolates were obtained by either method. All of the serotype O:8 isolates were virulent to mice, causing the death of adults after oral challenge. This is the first report that associates Y. enterocolitica serotype O:8 with a natural reservoir.