Porcine Protein Research Articles

Immunoreactive pancreatic colipase, lipase and phospholipase A 2 in human plasma and urine from healthy individuals

A radioimmunoassay for each of the human pancreatic proteins (colipase, lipase and phospholipase A 2 is described. Determinations of the mean concentration of each protein in plasma and urine from healthy individuals were carried out with the radioimmunoassays. The values obtained in plasma were 0.5 nM (5.3 μg/l), 0.6 nM (32 μg/l) and 0.3 nM (4.3 μg/l) for colipase, lipase and phospholipase A 2, respectively. In urine, the corresponding values were found to be 0.2 nM (2.4 μg/l), 0.09 nM (4.4 μg/l) and less than 0.017 nM (0.2 μg/l). No physical interaction between any of the three proteins and the lipid particles of plasma was demonstrated by centrifugation experiments or gel filtration. Gel filtration of plasma depleted of fat by centrifugation showed the proteins only in their monomeric form. The corresponding porcine proteins displayed a binding to antibodies against the human proteins, but with a lower affinity than the homologous interactions. The binding was weak but could differentiate between the porcine proforms and activate ones, i.e., procolipase and colipase 87.

Two dimensional double-quantum and COSY spectra of porcine adenylate kinase.

1H double-quantum and COSY NMR-spectra of the porcine protein adenylate kinase have been obtained in order to assign aromatic spin systems within this protein, which contains seven tyrosyl, five phenylalanyl, and two histidyl residues. Double-quantum spectroscopy in its simple form allowed the assignment of five connectivities in the aromatic region and therefore proved inferior to COSY, which allowed the assignment of connectivities within nine aromatic residues.

Size Exclusion/HPLC of Heated Water Soluble Bovine and Porcine Muscle Proteins

ABSTRACTProteins (280 nm absorbance) in water extracts from bovine semi‐membranosus and porcine longissimiu were separated by size exclusion (SEC) high performance liquid chromatography(HPLC). SEC/HPLC profiles showed six heat sensitive fractions ≥ 10,000 daltons. These fractions ranged from peak 1 at 375,800 daltons in bovine and 170,000 daltons in porcine down to myoglobin, peak 6, at 15,200 and 19,500 daltons for bovine and porcine, respectively. SEC/HPLC showed the loss of certain peaks at specific time/temperature treatments. Heat labile porcine proteins ≥ 10,000 daltons represented about 41.5 area percent of the total extractable components after heating to 60°C. In bovine, this represented about 37.5 area percent. In both species, heating at 70°C for 30 min or at 75°C for 0 min resulted in protein solubility loss to about 1 area percent or less.

Extrusion studies of mixtures containing certain meat offals: Part 1—Objective properties

A study was made of thermoplastically extruded products prepared from soy grits incorporating varying percentages of offal. Beyond 35% incorporation no satisfactory extrudate could be prepared. A comparison was therefore made of the expansion ratio, density and hydratability of extrudates prepared from mixes containing 20% and 35% of bovine or porcine offal with those containing soy grit alone. The offal sources consisted of untreated or alkali-extracted bovine and porcine lung and bovine tripe (rumen and reticulum) and bovine tripe extracted by sodium dodecyl sulphate (SDS). Under the determined optimum operating conditions, extrudates from all offal/soy mixtures had lower expansion ratios and rehydratability than those from soy grits alone. Nevertheless, mixtures containing 20% or 35% alkali-extracted offal protein expanded more (greatest expansion at 170°C), had lower density and greater hydratability than those containing these proportions of untreated offal or SDS-extracted offal protein. (The latter expanded most between 175–180°C.) Whereas the incorporation of SDS-extracted bovine or porcine lung protein failed to yield textured extrudates, the incorporation of SDS-extracted bovine tripe protein at the 20% level did so. Effects due to source were otherwise small.

NMR studies of myelin basic protein: X. Conformation of a determinant encephalitogenic in the rabbit

Residues 67 to 75 in myelin basic protein from several species comprise the sequence Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys that acts as an encephalitogenic determinant in the rabbit. Proton magnetic resonance spectra of human, bovine and porcine proteins display nulear Overhauser effects between δ-CH of Tyr-69 and the δ-CH 3 of Leu-72, which indicate reverse-turn conformatons about the Gly-Ser residues. This effect occurs also in physiological saline solution at pH 6.0 but in dimethylsulfoxide solution the nuclear Overhauser effect disappears. Circular dichroism indicates that the protein when bound to ganglioside micelles acquires 30–40% α-helical conformation, but the reverse turn still persists in the sequence of the rabbit encephalitogen. These results suggest that the encephalitogenic region of the protein remains at the aqueous interface of the micelles.

Isolation and NH 2-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily : Immunocytochemical localization in pars distalis and pars nervosa of the pituitary and in the supraoptic nucleus of the hypothalamus

The isolation and purification of a 21,000-Da (p I 4.9) novel protein from porcine anterior pituitary and whole human pituitary is described. Comparison of the NH 2-terminal sequence of the first 77 and 81 residues of the human and porcine homologs shows only one conservative substitution at residue 12, namely an Ala for a Thr between these two species. Such high sequence homology is also reflected in their amino acid composition. A computer data-bank search using a mutation data matrix and comparison with 338,327 segments of proteins revealed that this substance should be classified as belonging to a new protein superfamily. Immunocytochemical staining, using an antibody produced against a synthetic fragment, revealed the presence of immunostainable material in the anterior and posterior lobe of the pituitary and in the supraoptic nucleus of the hypothalamus. No staining was observed in the intermediate lobe of the pituitary. Furthermore, purified neurointermediate lobe secretory granule preparations were also shown to contain this novel polypeptide.

Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

Purification of Two Heparin-Binding Proteins From Porcine Platelets and Their Homology With Human Secreted Platelet Proteins

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno-precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

Primary Structure and Molecular Evolution of Intestinal Calcium-binding Protein

Abstract The primary structure and the molecular evolution of protein were examined with bovine and porcine intestinal calcium-binding protein (ICaBP). The amino acid sequence of this protein was compared with that of ancestral calmodulin, which had been predicted as the most ancient form in the family of calcium-binding proteins.

Binding of porcine von Willebrand factor to human platelets in the presence of ristocetin

The interaction of porcine von Willebrand factor (vWF) with human platelets in the presence of ristocetin was examined. Binding was rapid, specific, saturable and dependent on ristocetin concentration with half-maximal stimulation occuring at a ristocetin concentration of 0.5mM. Assuming vWF to be a tetramer with a MW of 9.5×10 −5, approximately 94,000 vWF binding sites per platelet were found with an average binding constant of 2.1×10 −8M. Humam vWF competed with the porcine protein for this site only in the presence of ristocetin. Binding of porcine vWF to platelets was found to be less sensitive to reduction with dithiothreitol than platelet agglutinating activity.

Equine whey proteins

1. 1. Zone electrophoretic patterns of milk samples have been examined from 713 mares, and classified in terms of 5 anodic zones and 1 cathodic zone at pH 8.5 (NaOHH 3BO 3). 2. 2. Zone 1 protein exhibits genetic polymorphism, there being 5 variants, designated A–E. Zone 2 protein is the dominant whey protein and is designated a β-lactoglobulin. Zone 3 protein is equine serum albumin. Zone 4 contains equine α-lactalbumin and iron binding protein (probably transferrin). Zone 5 contains iron binding protein, probably lactoferrin. The cathodic zone 6 contains equine lysozyme. 3. 3. Methods of isolation and properties of these proteins are described and discussed. In particular, the amino acid sequence of the first 40 residues of equine β-lactoglobulin is compared with the corresponding sequences of the porcine and bovine proteins. The reason for the failure of peptide mapping to reveal the homology of the equine protein with other β-lactoglobulins is discussed.

A new acidic protein in porcine brain

An extremely acidic protein has been isolated in a purified form from porcine brain extract, by (NH 4) 2SO 4 fractionation followed by column chromatography on DEAE-Sephadex A-50 and on Sephadex G-75. The purified protein was tentatively named as glutamic acid-rich protein because it was characterized by its remarkably high content of glutamic acid which accounted for 49% of the total amino acid composition. The protein appeared to be a single polypeptide chain with a molecular weight of 56 000–58 000, and had an isoelectric point of 4.6. The N-terminal amino acid sequence was Asp-Glu-Pro-Pro-Ser-GluGly. The immunochemical analysis using rabbit antiserum prepared to the porcine protein has suggested that it is present in the brain of human, cow, cat, dog and goat as well as in various goat organs including liver, kidney, heart, small intestine and spleen.

High levels of a calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein

Sperm from hamster, human, rooster, rabbit and sea urchin were found to contain relatively high levels of calcium-dependent modulator protein. Using rabbit sperm the modulator protein was found to be exclusively located in the sperm head fraction (nuclei + acrosomes) with no activity present in the midpiece or tail regions. The modulator protein represents approximately 12% of the total soluble protein found in the sperm head fraction and is similar to porcine and brain modulator proteins in its ability to activate brain cyclic nucleotide phosphodiesterase, its heat stability and electrophoretic migration. We have also observed modulator protein to be present in high levels in sea urchin eggs.

Turnover of tubulin and the N site GTP in chinese hamster ovary cells

Radioactively labeled tubulin from Chinese hamster ovary (CHO) cells can be isolated by copolymerization with nonradioactive porcine brain microtubule protein. 75% of the soluble tubulin in CHO extracts co-polymerizes with the porcine protein through several cycles, without preferential loss of either CHO or porcine subunits. After phosphocellulose chromatography of the co-polymerized microtubules, the CHO tubulin is radiochemically homogeneous, as judged by SDS-polyacrylamide gel electrophoresis. CHO tubulin purified in this way has 1 mole of nucleotide per mole of protein noncovalently bound at the non-exchangeable or N site. Thin-layer chromatography indicates that the N site nucleotide is entirely ribo-GTP. Label and chase experiments show that the N site GTP exchanges intracellularly with a half-time of 33 hr in growing cells which have a generation time of 17 hr, while the tubulin poly-peptides are degraded with a half-time of 48 hr. Intracellular hydrolysis of the γ-phosphate of the N site nucleotide can be detected but occurs very slowly, with a half-time of 24 hr. These results suggest that the N site nucleotide may function in vivo as a stable structural co-factor of the tubulin molecule and render improbable the possibility that it has a regulatory role in microtubule assembly.

Immunological results of hepatic assistance procedures

Hemodynamic and immunological effects were studied in eight normal calves during and after “hepatic assist” with a pig liver. Use of an oxygenator in the circuit allowed control of the rate of perfusion of the liver and using this system no hypotension or metabolic acidosis was observed in the recipient. Porcine protein was present in the sera of six surviving calves up to 14 days after perfusion. No antibodies to this protein were demonstrated either by immunodiffusion or by tanned red cell agglutination. Hypersensitivity and anaphylaxis did not occur when the calves were challenged intravenously with poreine protein and pig liver extract 3 weeks after the initial perfusion. No antibodies were demonstrated in the calf either as a primary response to circulating pig antigen or as a secondary response to a challenge with porcine protein or pig liver extract. Possible reasons for these findings and their significance are discussed.

Heterologous extracorporeal hepatic support: haemodynamic, biochemical, and immunological observations.

Abstract IN an attempt to provide an experimental appraisal of the value and limitations of extracorporeal heterologous liver perfusion as a method of temporary hepatic support, each of 4 normal calves was connected to a pig liver on two separate occasions at an interval of 1–3 weeks. With the technique employed these perfusions were tolerated for 6–7 hours. Acute liver failure was induced in another group of 5 calves by carrying out a portacaval anastomosis followed by ligation of the hepatic artery. Three of these animals were treated by repeated pig-liver perfusions. Survival was prolonged in 2 as compared with untreated controls, and the third made a complete recovery. Porcine protein was detected in all the calves after perfusion but antibody to this protein was only detected in 2 calves. Skin-grafts taken from the liver-donor pig during the second or third perfusion were accepted by the recipient calves for much longer periods than did unperfused animals. When 3 long-term surviving calves were challenged with intravenous porcine plasma only mild and transient reactions were seen. It is suggested that extracorporeal liver perfusion is beneficial, it is relatively free from immunological reactions, and that this procedure appears to confer on the recipients some degree of immunological tolerance.

On comparison of interactions of a "bitter-sensitive protein" from porcine tongues with human taste thresholds.

Doig, Lopiekes and I earlier reported the interactions of a porcine protein fraction with bitter compounds1. Price has recently commented on the poor agreement of our data with, as he states, “characteristics which might be expected of ‘the bitter-receptor protein’”2. I would like to make the following comments on comparisons drawn between porcine association constants and human bitter-threshold values.

Electrophoretic and Immunoelectrophoretic Analysis of the Proteins in the Sow’s Mammary Secretions throughout Lactation

Lactation in the sow can be separated into 3 stages, based on the relative concentrations of 3 electrophoretic whey proteins in the mammary secretions: (1) parturition (approximately 72 hours); (2) midlactation (approximately third day to sixth week); (3) late lactation (approximately sixth week to dryness). The concentration of protein (18 per cent), and dry matter (28 per cent), in the secretion at parturition, decreases rapidly to its lowest levels during midlactation (protein, 5 per cent; dry matter, 16 per cent). In late lactation there is a gradual increase to approximately 10 per cent protein and 24 per cent dry matter. Whey proteins accounting for 80 per cent of the total protein at parturition rapidly decrease to 50 per cent, at which level they remain until midlactation. Around the fifth or sixth week of lactation, casein increases to approximately 70 per cent of the total protein, and this relationship persists to the end of the lactation. Using immunoelectrophoretic techniques, only 1 of the major electrophoretic fractions of the sow’s mammary secretions (SMS) appeared to be exclusively a whey protein. The major antigens of the remaining electrophoretic fractions were serologically related to porcine serum proteins. Antibodies to casein, a ‘milk protein’ by definition, could not be demonstrated in the antisera used in this study. Although the quantitative changes in SMS whey proteins are pronounced at the various stages of lactation, the kinds of protein as expressed by im munoelectropherograms appear in the main unchanged.

The Effect of Complements on Soluble Antigen-Antibody Complexes

Summary The ability of complement from various animal species to precipitate soluble rabbit antigen-antibody complexes in the region of antigen excess has been demonstrated. Guinea pig and rabbit complements precipitated the soluble complexes better than human or pig complements, and guinea pig complement precipitated the complexes better than rabbit complement. The failure of pig and human complements to precipitate soluble complexes to the same extent as guinea pig and rabbit complements may be partially caused by several factors: (1) an inhibition resulting from cross reactions between the antibodies in the complexes and the human and porcine proteins, (2) the presence of an inhibitory factor which was found to be inactivated upon aging the serum, and (3), the physical and chemical characteristics of complement molecules themselves. Two possible hypotheses were set forth to describe the mechanism involved in the precipitation of soluble complexes by complement. These hypotheses were: (1) complement fixed to the complexes to such an extent that the increase in weight and/or neutralization of charges caused the complexes to precipitate, and (2), the complement was multivalent to antibody and/or antigen and, therefore, was able to link the complexes together to form precipitating aggregates. The ability of guinea pig and human complement to cause precipitation of “univalent” (co-precipitating) antigen-antibody complexes was demonstrated.