Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.     

Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.     

Purification and partial sequence analysis of insulin-like growth factor-1 from bovine colostrum.

Growth-promoting activity in bovine colostrum has been detected as the capacity to stimulate protein synthesis in L6 myoblasts. By using this assay as a measure of bioactivity, a growth factor has been purified to near homogeneity from centrifuged colostrum by a series of steps including acid extraction, chromatography on sulphopropyl-Sephadex, followed by adsorption to, and elution from, C18 columns using acetonitrile and propan-1-ol gradients. The purified growth factor has a low solubility at neutral and alkaline pH and has an Mr of 7800 by gel-permeation chromatography. Sequence analysis of the first 30 amino acids from the N-terminus indicated complete identity in this region with human insulin-like growth factor-1. Accordingly we conclude that the purified growth factor is bovine insulin-like growth factor-1.     

Occurrence of a unique sialyl tetrasaccharide in colostrum of a bottlenose dolphin (Tursiops truncatus)

Crude oligosaccharides were recovered from bottlenose dolphin (Tursiops truncatus) colostrum after chloroform/methanol extraction of lipids and protein precipitation, and purified using gel filtration, anion exchange chromatography and high performance liquid chromatography (HPLC). Their chemical structures characterized by NMR spectroscopy were as follows: GalNAc(beta1-4)[Neu5Ac(alpha2-3)]Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc and Gal(alpha1-4)Gal(beta1-4)Glc. The monosialyltetrasaccharide and neutral trisaccharide have not previously been found as free forms in any natural sources including milk or colostrum, although these structures have been found in the carbohydrate units of glycosphingolipids GM2 and Gb3.     

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.     

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactivate up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145 000 to 172 000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.     

The carbohydrate linkage site of cow colostrum trypsin inhibitor

Cow colostrum trypsin inhibitor (chromatographic form AIV[7])was subjected to basic conditions that favour beta-elimination of carbohydrates from O-glycosidic linkages. The unchanged carbohydrate composition and the unchanged values for serine and threonine indicate the presence of an alkali-stable N-glycosidic bond to asparagine. From a tryptic digest of S-aminoethylated inhibitor the glyco-decapeptide (residues 24 through 33) was isolated. The carbohydrate composition was identical with that of the S-aminoethylated inhibitor. Further degradation of this peptide by carboxypeptidase C (and aminopeptidase) produced the glycopeptide Asn(CHO)-Ser-(Thr) with the same carbohydrate composition. Thus, a single carbohydrate moiety is bound by a N-glycosidic linkage to asparagine-27 of the colostrum inhibitor. It is located opposite to the reactive site at the base of a pear-shaped molecule.     

Bovine fetus-specific serum proteins. Purification and characterization of alpha1-fetoprotein and immunological identification of alpha2- and beta-fetoproteins.

Bovine alpha1-fetoprotein was isolated from fetal calf serum by successive procedures of concanavalin A-Sepharose chromatography, DEAE-Sephadex chromatography, SP-Sephadex chromatography and preparative disc polyacrylamide gel electrophoresis. The bovine alpha1-fetoprotein preparation was considered homogeneous by several physicochemical and immunochemical criteria. Bovine alpha1-fetoprotein has a molecular weight of 68 000 with an amino acid composiotn similar to that of other mammalian alhpa1-fetoprotein. In addition, bovine alpha1-fetoprotein was shown to exist as two distinct variants on the basis of carbohydrate heterogeneity. alpha2-Fetoprotein and a new beta-fetoprotein were immunologically identified in fetal calf serum. These fetoproteins, like alpha1-fetoprotein, were not detectable in non-pregnant cow serum by immunoelectrophoresis.     

Isolation of tri-antennary enriched and tri-antennary depleted alpha-1-protease inhibitor

Alpha-1-protease inhibitor, (alpha-1-PI), the major inhibitor of serine proteases in human plasma, has three asparagine-linked carbohydrate chains located at positions 46, 83 and 247. The protein has a microheterogeneity which is seen on isoelectric focusing and which is a result of whether the various carbohydrate chains are in bi- or tri-antennary forms. Tri-antennary enriched forms of alpha-1-PI are associated with inflammation. By using a combination of three methods, reductive salting out, Sepharose-bound Concanavalin A affinity chromatography, and Sepharose-bound anhydrochymotrypsin, biologically active alpha-1-PI was obtained in tri-antennary enriched and tri-antennary depleted forms. These preparations should be useful for studies on the physiological role of the carbohydrate moiety in alpha-1-PI.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Human leukocyte granule elastase: rapid isolation and characterization.

Human granulocytic elastases have been purified by a two-step procedure involving affinity chromatography of crude extracts of leukocytic granules on Sepharose-Trasylol, followed by ion-exchange chromatography on CM-cellulose to resolve the isoelastases. All of these enzymes were found to be glycoproteins with the carbohydrate content of the major form being composed essentially of only neutral sugars. The molecular weight of this form was found to be near 30 000 daltons with the other forms being slightly higher. Preliminary structural analyses indicate that all of the elastase isozymes have identical NH2-terminal sequences suggesting that the differences in mobility of the four proteins are not due to different degrees of activation from a common zymogen but, more likely, from minor changes in carbohydrate content. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase, but both are inhibited by synthetic elastase active-site directed low molecular weight compounds (Tuhy, P. M., and Powers, J. C. (1975), FEBS Lett. 50, 359) as well as by plasma alpha-1-proteinase inhibitor (formerly called alpha-1-antitrypsin). In the latter case a stable complex with mol wt of 78 000 daltons is formed indicating the formation of a 1:1 complex.     

Identification and characterization of apelin peptides in bovine colostrum and milk by liquid chromatography-mass spectrometry

Apelin peptides were recently identified as endogenous ligands of the APJ receptor. It has been hypothesized that these peptides are initially provided to the newborn by nursing and might be involved in gastrointestinal tract development. As apelin peptides may have different effects on the APJ receptor as a function of their size, knowledge of their exact structure in early milk is essential to clarify their action in gastrointestinal tract development. Bovine colostrum is thought to contain high concentrations of a wide diversity of apelin peptides, but none of them have yet been rigorously characterized. To identify and monitor apelin peptides in bovine colostrum, we developed a cation exchange extraction step followed by untargeted liquid chromatography coupled to high resolution and high mass accuracy mass spectrometry (LTQ-Orbitrap). Using this approach, we characterized 46 endogenous apelin peptides in bovine colostrum, which varied in relative abundance from one colostrum to another. Mature as well as commercial milk samples were also studied. Taken together, our data demonstrate that the multiplicity and variability of apelin peptides are biologically relevant and change during milk maturation to reach a more constant composition in mature milk.     

Heterogeneity of the basic pancreatic inhibitor (Kunitz) in various bovine organs

Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test.     

Isolation and characterization of multiple forms of ovine pancreatic deoxyribonuclease. Chromatograhpic behavior of the enzyme on concanavalin A-agarose and carboxymethylcellulose columns.

A new procedure has been devised for the purification of ovine DNase, including (NH/4)2SO4 fractionation, two steps of CM-cellulose chromatography, concanavalin A-agarose chromatography, and gel filtration on Sephadex G--100. The enzyme, like bovine DNase, exhibits multiplicity due to changes in the primary structure and the sugar structure of the carbohydrate moiety. Unlike bovine DNase, ovine DNase does not have sialic acid in any of its multiple forms. Concanavalin A-agarose is useful in the purification of not only ovine but also bovine DNase. For ovine DNase, it is a necessary and key step of purification; for bovine DNase, it can be used to purify commercial preparations of DNase free from proteases in a single step as judged by its stability in Ca2+-free media at pH 8.0. The purified enzyme has a specific activity equal to that of a highly purified DNase and presumably contains predominantly DNases A and C. Two of the four forms of ovine DNase have been purified to apparent homogeneity and subjected to chemical analysis. The present results show that bovine and ovine DNases have indistinguishable molecular weights and identical end groups, suggesting that they may have the same number of amino acid residues. The amino acid composition indicates that two enzymes may have six residues of amino acids subject to substitution which can be explained by single base changes in their genetic code words. Amino acid analyses also indicate that the most likely difference between two forms of ovine DNase is the substitution of Leu for Arg.     

Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.

The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.     

Purification and characterization of an inhibitor protein with cytochalasin-like acitvity from bovine adrenal medulla

A protein preparation with cytochalasin-like activity has been obtained from bovine adrenal medulla. Analysis by electrophoresis in SDS-polyacrylamide gels and chromatography in a Sephacryl S-200 column indicated that the inhibitor activity coincided with a 90 000 dalton polypeptide. The inhibitor decreased high-affinity binding of [³H]cytochalasin B to actin nuclei, apparently by competing with the drug for thesame binding site. At substoichometric levels, the inhibitor had a potent effect on actin filament elongation and on actin-dependent gelation of cell extracts in vitro. These results suggest that the inhibitor may be involved in the control of actin filament assembly and interaction in the adrenal medulla.     

Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in human and bovine milk

Two hypothalamic peptide hormones, luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH), have been isolated from human milk and bovine colostrum. Acidified methanolic extracts, prepared from human milk, bovine colostrum and rat hypothalami, as well as synthetic LHRH and TRH markers were subjected to high-pressure liquid chromatography (HPLC). The eluates were tested for the presence of LHRH and TRH by specific radioimmunoassays. It was found that milk extracts contain significant amounts of LHRH (3.9 - 11.8 ng/ml) and TRH (0.16 - 0.34 ng/ml), which comigrate with the corresponding marker hormones and with those of hypothalamic origin. The HPLC-purified LHRH from both human and bovine milk was bioactive in a dose-response manner similar to synthetic LHRH.     

Polypeptide heterogeneity of hamster and calf fibronectins.

The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].     

Branch specificity of bovine colostrum and calf thymus UDP-Gal: N-acetylglucosaminide beta-1,4-galactosyltransferase

The branch specificity of bovine colostrum and calf thymus UDP-Gal:N-acetylglucosaminide beta-1----4-galactosyltransferase toward several branched oligosaccharides, which form part of the complex-type N-glycans of glycoproteins, was investigated. A novel method was used based on acetolysis of the bi[14C,3H] galactosylated oligosaccharide products formed by the enzymes in vitro and analysis of the acetolysis fragments by high-pressure liquid chromatography. It could be established that the galactosylation of different oligosaccharide branches occurred in a preferred order. No difference in branch specificity was observed between the soluble bovine colostrum galactosyltransferase and the enzyme that had been solubilized from calf thymus membranes. A preferential pathway for the biosynthesis of bisialylated biantennary glycans is proposed.     

Demonstration and partial purification of lactoperoxidase from human colostrum

A peroxidase with stability, chromatographic and immunoreactive properties similar to that of bovine lactoperoxidase has been partly purified from human colostrum. Hydrophobic interaction chromatography on Phenyl-Sepharose C1-4B gave a 10-fold purification with an apparent recovery of about 45%. The enzyme was quantitatively and specifically adsorbed to beads of anti-lactoperoxidase (bovine)-Protein A-Sepharose. No adsorption of the enzyme was observed on immunoadsorbent columns prepared with high-titre polyclonal antibodies raised against human myeloperoxidase and human eosinophile peroxidase.