Absorption and retention of free and milk protein-bound cyano- and hydroxocobalamins. An experimental study in rats.

INTRODUCTION: Cobalamin/Vitamin B12 (Cbl) is an essential vitamin, supplied mainly as hydroxocobalamin (OHCbl) by animal products, including cows' milk. Cyanocobalamin (CNCbl) is the usual form in vitamin pills. The aim was to explore absorption and tissue accumulation of two Cbl forms, administered alone or bound to milk protein. MATERIALS AND METHODS: We synthesized labeled OH[(57)Co]Cbl from commercially available CN[(57)Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250-300 g) received labeled Cbl by gastric gavage. First, we administered CN[(57)Co]Cbl, free or rbTC-bound (n = 15 in each group). Rats were sacrificed after two, 24, and 48 h. In the following studies, rats were sacrificed after 24 h. We compared absorption of free or rbTC-bound CN[(57)Co]Cbl added to cows' milk and analogous absorption of OH[(57)Co]Cbl, free or rbTC-bound, to absorption of free CN[(57)Co]Cbl, (n = 10 in each group). Blood, tissues, 24-h urine and feces were collected. Labeled Cbl was measured using a gamma counter. Results are expressed as percentage of administered dose. RESULTS: Absorptions of CNCbl and OHCbl were neither influenced by rbTC-binding nor administration in milk. Absorption increased in the first 24 h with no further tissue accumulation during the subsequent 24 h. Accumulation of free CNCbl and (OHCbl) was 1.4, (4.1) (liver); 20.2, (16.4) (kidney); and 0.05, (0.02) (plasma)% 24 h after administration. Total organ accumulations were 21.6, (20.5)%. While total accumulations of CNCbl and OHCbl were equal, distributions between liver, kidney, and plasma showed significant differences (p < 0.0001; p = 0.01; p < 0.0001). CONCLUSIONS: Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated.

Preparation and comparison of cytotoxic complexes formed between oleic acid and either bovine or human α-lactalbumin.

α-Lactalbumin is a ubiquitous calcium-binding milk protein with a well-characterized function in regulating the synthesis of lactose. An entirely different activity has been shown to occur when a complex is formed between calcium-free α-lactalbumin and oleic acid. This complex shows strong cytotoxic action against several cancer cells, and several mechanisms have been suggested to account for this cell-killing activity. Most studies have been performed using the human protein, but bovine α-lactalbumin shows similar activity. A new and simple 2-step method for purification of calcium-free α-lactalbumin has been developed, and the resulting highly purified preparation was used to generate a complex with oleic acid. Using 3 different cell lines and 2 types of cell viability assays, the bovine and human α-lactalbumin showed comparable cytotoxic activity. The effect was apparent after 15 min of incubation and was inhibited by the presence of fetal bovine serum or bovine serum albumin. The bovine protein might be a useful alternative to the human protein, but also raises the question whether cytotoxic activity could be generated in different kinds of food containing α-lactalbumin.

Phosphatidylserine exposure and procoagulant activity in acute promyelocytic leukemia.

BACKGROUND: Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown. METHODS: Lactadherin, a milk protein with stereospecific binding to phosphatidyl-L-serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays. RESULTS: Plasma procoagulant activity of NB4 and APL cells increased approximately 15-fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As(2)O(3). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As(2)O(3) and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80-85% of intrinsic FXase, FVIIa-tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure. CONCLUSIONS: PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure.

BACKGROUND: Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. OBJECTIVE: To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo. METHODS: Lactadherin, a PS-binding milk protein, was utilized together with annexin V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. RESULTS: The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin V required a threshold of 2.5-8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin V binding at physiologic Ca(2+) concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. CONCLUSIONS: In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.

Plasmin digestion of photooxidized milk proteins.

Plasmin-mediated hydrolysis of 6 different milk protein preparations [alpha(S)-casein (alpha(S1) + alpha(S2)), beta-casein, kappa-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of alpha(S)-casein, consisting of a mixture of alpha(S1)- and alpha(S2)-casein and a preparation of beta-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.

Inhibitory activities of bovine macromolecular whey proteins on rotavirus infections in vitro and in vivo.

Rotavirus is a major cause of infantile viral gastroenteritis and can lead to severe and sometimes lethal dehydration. Previous studies have shown that breast-fed children are better protected against symptomatic infections, and that the milk fat globule protein lactadherin might be at least partly responsible for this effect. In vitro studies have shown that human lactadherin, in contrast to the bovine ortholog, could inhibit rotavirus infectivity, and that bovine MUC1 and a commercially available bovine macromolecular whey protein (MMWP) fraction proved to be effective. The present work describes the versatility of MMWP against the infection of 2 human intestinal cell lines (Caco-2 and FHs 74 Int) by 4 different rotavirus strains (Wa, RRV, YM, RF). Isolation of a protein fraction (CM3Q3) from MMWP that effectively inhibits rotavirus infectivity in vitro is documented. Purification was achieved by monitoring the rotaviral inhibitory activity in fractions obtained from 2 consecutive steps of ion-exchange chromatography. The major component of CM3Q3 was shown to be bovine IgG, and the attenuating capacity of this fraction is most properly linked to this component. The capacity of MMWP, MUC1, lactadherin, and the CM3Q3 fraction to inhibit the infectivity of the murine EMcN rotavirus strain was analyzed in adult BALB/c mice by using 2 different amounts of virus (10 and 100 times more than 50% the viral shedding doses). Only CM3Q3 was able to significantly affect the shedding of rotavirus in the stools of experimentally infected mice when the high viral dose was given. Detection of rotavirus-specific serum antibodies showed that the high dose infected all groups of mice. Experiments with the low dose of virus implied that all the tested milk proteins could affect the viral shedding in stools; in addition, use of MUC1, MMWP, and CM3Q3 prevented the appearance of serum viral antibodies. The advantages of using bovine immunoglobulins to induce passive immunity against rotavirus have been substantially investigated, although studies have mainly focused on the use of derivatives from immunized cows, especially colostrum. This report associates considerable activity against rotavirus infectivity with an ordinary whey product, suggesting that there might be alternatives to colostral-derived products.

Inhibitory effects of human and bovine milk constituents on rotavirus infections.

Among etiologic agents, rotavirus is the major cause of severe dehydration diarrhea in infant mammals. In vitro and in vivo studies have indicated that the human milk-fat globule protein lactadherin inhibits rotavirus binding and protects breast-fed children against symptomatic rotavirus infection. The present work was conducted to evaluate the effect of lactadherin, along with some other milk proteins and fractions, on rotavirus infections in MA104 and Caco-2 cell lines. It is shown that human, and not bovine, lactadherin inhibits Wa rotavirus infection in vitro. Human lactadherin seems to act through a mechanism involving protein-virus interactions. The reason for the activity of human lactadherin is not clear, but it might lie within differences in the protein structure or the attached oligosaccharides. Likewise, in our hands, bovine lactoferrin did not show any suppressive activity against rotavirus. In contrast, MUC1 from bovine milk inhibits the neuraminidase-sensitive rotavirus RRV strain efficiently, whereas it has no effect on the neuraminidase-resistant Wa strain. Finally, a bovine macromolecular whey protein fraction turned out to have an efficient and versatile inhibitory activity against rotavirus.

Thermal inactivation kinetics of bovine cathepsin D.

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion.

A novel heparin-binding protein was purified to homogeneity from bovine prepartum mammary gland secretion using heparin-Sepharose chromatography and reverse-phase high performance liquid chromatography successively. Structural information obtained by N-terminal amino acid sequencing of a series of proteolytically generated peptides permitted the cloning of the corresponding cDNA. The isolated cDNA was 1170 base pairs long and consisted of an 83-base pair 5'-untranslated region followed by a 702-base pair coding region and a 385-base pair 3'-untranslated region. The open reading frame resulted in a protein comprising 234- amino acid residues, including a signal sequence. Instead of Lys(24) as the predicted N terminus, Edman degradation of the native protein revealed N-terminal processing at two sites as follows: a primary site between Arg(31)-Gly(32) and a secondary site between Arg(51)-Ser(52). The amino acid sequence showed a significant similarity with that of human (60%) and mouse (53%) fibroblast growth factor-binding protein (FGF-BP). Accordingly, ligand blotting experiments revealed that bovine FGF-BP bound FGF-2. The theoretical mass of the protein predicted from the cDNA sequence is 22.5 kDa. However, the molecular mass of the purified protein was estimated to 28.6 kDa by mass spectrometry and 36 kDa by electrophoresis. The apparent molecular weight differences are most likely due to post-transcriptional modifications, shown to involve N- and O-glycosylation of Asn(155) and Ser(172), respectively. All 10 cysteine residues in the protein participated in disulfide bonds, and the pattern was identified as Cys(71)-Cys(88), Cys(97)-Cys(130), Cys(106)-Cys(142), Cys(198)-Cys(234), and Cys(214)-Cys(222). As the 10 cysteines of the three known FGF-BPs are positionally conserved, the disulfide bond pattern of bovine FGF-BP may be regarded as representative for the FGF-BP family.

A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms.

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein.

Very-low-density lipoprotein receptor (VLDLR) and alpha2-macroglobulin receptor/low-density-lipoprotein-receptor-related protein (alpha2MR/LRP) are multifunctional endocytosis receptors of the low-density lipoprotein receptor family. Both have been shown to mediate endocytosis and degradation of complex between plasminogen activators and type-1 plasminogen-activator inhibitor (PAI-1) by cultured cells. We have now studied the specificity of binding and endocytosis by VLDLR and alpha2MR/LRP among a variety of serine proteinase/serpin complexes, including various combinations of the serine proteinases urokinase-type and tissue-type plasminogen activators, plasmin, thrombin, human leukocyte elastase, cathepsin G, and plasma kallikrein with the serpins PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, alpha2-antiplasmin, alpha1-proteinase inhibitor, alpha1-antichymotrypsin, protease nexin-1, heparin cofactor II, and antithrombin III. Binding was estimated with radiolabelled ligands in ligand blotting analysis and microtiter well assays. Endocytosis was estimated by measuring receptor-associated protein (RAP)-sensitive degradation of radiolabelled complexes by Chinese hamster ovary cells transfected with VLDLR cDNA and by COS-1 cells, which have a high endogenous expression of alpha2MR/LRP. We found that the receptors bind with high affinity to some, but not all, combinations of plasminogen activators and thrombin with PAI-1, protease nexin-1, protein C inhibitor, and antithrombin III, while complexes of many serine proteinases with their primary inhibitor, i.e. plasmin/alpha2-antiplasmin complex, do not bind, or bind with a very low affinity. Both the serine proteinase and the serpin moieties contribute to the binding specificity. The binding specificities of VLDLR and alpha2MR/LRP are overlapping, but not identical. The results suggest that VLDLR and alpha2MR/LRP have different biological functions by having different binding specificities as well as by being expressed by different cell types.

Breast carcinoma epithelial cells express a very low-density lipoprotein receptor variant lacking the O-linked glycosylation domain encoded by exon 16, but with full binding activity for serine proteinase/serpin complexes and Mr-40,000 receptor-associated protein.

Very-low density lipoprotein receptor (VLDLR) belongs to the low-density lipoprotein receptor family of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr-40,000 receptor-associated-protein (RAP), and some serine proteinase/serpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with Mr 105,000 and Mr 130,000, respectively [Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjøller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. & Andreasen, P. A. (1995) J. Biol. Chem. 270, 20,855-20,869]. We now demonstrate that these two forms correspond to forms with the absence (type-II) and presence (type-I) of the O-linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrate that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell-surface-associated serine proteinase/serpin complexes in breast carcinomas.

Plasminogen activation system in human milk.

BACKGROUND: Plasmin is the major endogenous protease present in milk. The level of plasmin activity is controlled by the availability of the precursor plasminogen and by the levels of plasminogen activators and inhibitors. Recently, a differential distribution of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) has been demonstrated in bovine milk. To assess whether this distribution pattern is a general feature, the occurrence of components of the plasminogen activation system in different fractions of human milk was investigated. METHODS: Milk samples were separated into the following fractions; milk fat, skim milk, and milk cells by centrifugation. The different fractions were detected for the presence of plasminogen and plasminogen activators by immunoblotting and zymography. The distribution of t-PA and u-PA was investigated by ligand binding analysis. t-PA-catalyzed plasminogen activation was examined by a coupled chromogenic assay. RESULTS: A differential distribution of plasminogen, t-PA, and u-PA was found. Casein micelles were found to exhibit t-PA and plasminogen binding activity, whereas the u-PA receptor was identified as the u-PA binding component in the cell fraction. Furthermore, human casein enhanced t-PA-catalyzed plasminogen activation, comparable to the enhancing effect obtained with fibrinogen fragments. CONCLUSION: The finding of a differential distribution of u-PA and t-PA in milk suggests that the two activators may have different physiological functions, which involve protection against invading microorganisms and maintenance of patency and fluidity in the ducts of mammary gland, respectively.

Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas.

We have investigated the localization of urokinase-type plasminogen activator (u-PA), type-1 plasminogen-activator inhibitor (PAI-1), u-PA receptor (u-PAR) and alpha(2)-macroglobulin- receptor/low-density-lipoprotein-receptor-related protein (alpha(2)MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of 133 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph-node metastases were stained with monoclonal antibodies. Formalin-fixed sections of 15 primary tumors and 2 lymph-node metastases were stained with polyclonal antibodies. In primary tumors, u-PA and PAI-1 immunoreactivities were intense in macrophages and mast cells, and moderate in benign and malignant epithelial cells as well as in myofibroblasts and endothelial cells. A sub-group of poorly differentiated tumors showed particularly strong staining of stromal fibroblasts. u-PA immunoreactivity was also present in lymphocytes. alpha(2)MR/LRP and u-PAR immunoreactivities were intense in macrophages, but apart from these cells, alpha(2)MR/LRP was found only in fibroblasts, and u-PAR only in tumor cells located peripherally in tumor-cell clusters and glands and some myofibroblasts in the adjacent stroma. Lymph-node metastases showed staining for u-PA and PAI-1 both of cancer cells and of stromal fibroblasts, also staining for u-PA of lymphocytes. Similarly to some of the poorly differentiated primary tumors, approximately half of the metastases showed very strong staining of stromal fibroblasts, and extracts of these metastases had higher u-PA and PAI-1 levels, as determined by ELISA, than extracts of metastases without this staining pattern. alpha(2)MR/LRP was present only in fibroblasts and u-PAR only in some tumor cells. The presence of u-PA, PAI-1, alpha(2)MR/LRP and u-PAR was controlled biochemically by immunoblotting analyses, ligand-blotting analyses, and direct and reverse zymography. The spatial distribution and the variation in concentration of the various components of the plasminogen-activation system point to a complex, multifunctional role for the 4 proteins in and/or during the development and spread of breast cancer.

Very low density lipoprotein receptor binds and mediates endocytosis of urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex.

Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.

Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of M(r) 40,000 receptor associated protein.

We here report that the M(r) 40,000 receptor associated protein (RAP), previously found to bind to alpha 2-macroglobulin receptor/low density lipoprotein receptor related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330), binds to an M(r) 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N-terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL-R). Experiments with the human mamma carcinoma cell line MCF-7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL-R, similarly to alpha 2MR/LRP and gp330.

The plasminogen activation system in bovine milk: differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor.

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of kappa-casein and dimeric alpha s2-casein were identified as t-PA binding components. The kappa-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229-236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor.

The latent form of type-1 plasminogen activator inhibitor (PAI-1) acquires inhibitory activity by denaturation followed by refolding. We show here that the reactions of denatured/refolded PAI-1 with plasminogen activators are affected by low concentrations of SDS, which may remain after using SDS for denaturation. Without SDS, the active fraction of denatured/refolded PAI-1 comprised around 60%. Increasing SDS concentrations led to conversions to an inert form without inhibitory activity; then to a substrate form, that is being cleaved proteolytically in the reactive centre by the activators without complex formation, and finally to a second inert form. The first two conversions were associated with changes of the reactivity with monoclonal antibodies and of the thermal stability, respectively. Our results define clearly different interconvertible forms of denatured/refolded PAI-1, distinguish these from the latent and the reactive-centre-cleaved forms, and provide conditions for reproducibly producing reactive-centre-cleaved PAI-1 and PAI-1/activator complexes.

Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies.

Type-1 inhibitor of plasminogen activators (PAI-1) occurs in purified preparations in a latent form that can be activated with denaturants; in vivo, latency is prevented by binding to vitronectin. We have compared latent, denaturant-activated and reactive centre-cleaved human PAI-1 with respect to thermal stability and affinity to monoclonal antibodies. By both criteria, latent and cleaved PAI-1 are very similar or indistinguishable, and clearly different from active PAI-1. Our findings suggest that the conformations of latent and reactive centre-cleaved PAI-1 are similar and resemble the so-called relaxed (R) serpin conformation, while that of active PAI-1 is different and resembles the stressed (S) serpin conformation.