Proteolysis of milk fat globule membrane proteins in preterm milk: a transient phenomenon with a possible biological role?

Milk fat globule membrane (MFGM) proteins constitute a milk fraction currently of great interest, as they appear to significantly contribute to milk protective role. We investigated these proteins in human preterm colostrum and milk. For the former we found a peculiar 2-DE pattern, with a spot concentration at low molecular weight, which mass spectrometry analysis showed to be fragments belonging to some MFGM proteins with a well-known biological and especially immunological role: lactadherin, membrane-associated lactoferrin, butyrophilin, clusterin and heavy-chain immunoglobulin. Since we were able to rule out protease activity after specimen collection, we hypothesize the localization of the proteolytic enzymes in the alveolar cell membranes of the mammary gland. This mechanism is probably under hormonal control and the unexpected advent of preterm delivery would not allow hormonal conditions typical of lactation to occur immediately, causing a delay in enzymatic inhibition. This hypothesis is supported by some of our results, picturing a peculiar transient phenomenon of adaptation of the mammary-gland-membrane proteins after preterm delivery. Further studies will be required to verify whether the presence of protein fragments exerts a specific biological and immuno-defensive role in preterm infants, thus adding evidence to the outstanding biological role and benefits of mother's own milk in feeding preterm infants.

Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus galloprovincialis. cavalett@unipmn.it.

The pro-oxidant effect of H2O2 at a concentration of 20 microM was examined in the digestive gland of Mytilus galloprovincialis, a bivalve mollusc frequently used in biomonitoring programs. The oxidative stress caused by H2O2 has been evaluated in terms of lipid peroxidation and lysosomal system alteration. Complex cellular antioxidant defence mechanisms of the mussel were investigated at the enzymatic and non-enzymatic level in order to explain their relative role in reducing the risk of oxidative injury. Metallothionein, glutathione, superoxide dismutase, catalase and glutathione peroxidase were assayed after 1, 4 and 7 days of exposure to H2O2. The metallothionein content showed an increase by 43% after 4 days of exposure, followed by a decrease back to control values at 7 days. Antioxidant enzyme activities followed a similar pattern with a moderate increase after 1 or 4 days of treatment and a return to control values at 7 days. All data indicate a 'transient' oxidative stress response, after which mussel cells restore the redox balance.

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.

Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae.

Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis.

Metallothionein (MT) is a sulfhydryl-rich protein involved mainly in heavy metal homeostasis and detoxification. In this study, the use of the mussel as an experimental model allowed us to test MT antioxidant properties at the molecular, cellular, and organism level. MT induction was achieved by mussel exposure to Cd (200 microg/l) in aquaria for 7 days followed by detoxification in the sea for 28 days. Cd-preexposed and nonexposed mussels were then treated with Fe (300-600 microg/l) in aquaria for 3 days. Biochemical assays on digestive gland tissue showed that treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. The Cd-dependent resistance to oxidative stress was ascribed to MT induction, as Cd produced no significant variation of reduced glutathione and major antioxidant enzymes. Digital imaging of isolated digestive gland cells showed lower oxyradical rise and higher viability in cells from Cd-preexposed mussels after treatments with 0.5-5 mM H2O2. Analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. In conclusion, data suggest an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress.

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

Isolation and characterization of full and truncated forms of human breast carcinoma protein BA46 from human milk fat globule membranes.

We have isolated and characterized two proteins of 50 and 30 kDa from human milk fat globule membranes of healthy donors. N-terminal and internal sequencing revealed that the 50-kDa protein is the full-length human breast carcinoma protein BA46 that is highly expressed in human breast tumors. The 30-kDa protein is a truncated form of protein BA46 which consists of the C-terminal factor V/VIII-like domain of BA46 and which appears to anchor BA46 to the milk fat globule membrane. Defective release of the epidermal growth factor domain containing a surface RGD motif may be related to involvement of BA46 in breast cancer.

The unusual amino acid triplet Asn-Ile-Cys is a glycosylation consensus site in human alpha-lactalbumin.

Human alpha-lactalbumin has not been described as a glycoprotein, despite the fact that several alpha-lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the 45Asn in the usual triplet 45Asn-Gly/Gln-47Ser. We have found that human alpha-lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at 71Asn in the triplet 71Asn-Ile-73Cys, which is conserved in all known alpha-lactalbumins except red-necked wallaby. That a relatively small proportion of the protein is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of alpha-lactalbumin.

Absence in human milk of bovine beta-lactoglobulin ingested by the mother. Unreliability of ELISA measurements.

OBJECTIVES: To analyze the presence of bovine beta-LG in breast milk. METHODS: Human milk samples from 14 healthy non-atopic women on diets with different cow's milk contents were examined. The total concentration of beta-LG immuno-like proteins (beta-LGIP) was determined by enzyme linked immunosorbent assay (ELISA). Identification of antigens was done by N-terminal sequencing. RESULTS: beta-LGIP reactivity of the milk from subjects on different diets was not significantly different. Human lactoferrin, beta-casein and alpha-lactalbumin, were identified as cross-reacting antigens. CONCLUSIONS: False-positive results in ELISA determinations of bovine beta-LG in human milk might be due to cross-reactions between polyclonal antibodies and different protein antigens.

Human milk proteins may interfere in ELISA measurements of bovine beta-lactoglobulin in human milk.

It is widely believed that cow's milk proteins ingested by the mother, in particular beta-lactoglobulin (beta-LG), can pass into breast milk and thus sensitize predisposed infants. However, studies to evaluate bovine beta-LG in human milk have given conflicting results. The aim of this study was to analyse the correlation between the amount of cow's milk in the mother's diet and the presence of bovine beta-LG in breast milk. Human milk samples from 14 healthy non-atopic women on diets with different cow's milk contents were examined. The total concentration of bovine beta-LG or beta-LG immuno-like proteins (beta-LGIP) was determined by enzyme-linked immunosorbent assay (ELISA). Two separation procedures utilizing ELISA plates and an affinity chromatography column were set up to identify the human whey components recognized by the anti-beta-LG antibodies. beta-LGIP reactivities of milk from three groups on different diets were not significantly different. After splitting the antigen-antibody complexes, three main protein components, human lactoferrin, human beta-casein and human alpha-lactalbumin, were identified. This study would suggest that, at least in healthy subjects, false-positive results in ELISA determinations of bovine beta-LG in human milk might be due to cross-reactions between polyclonal antibodies and different protein antigens.

Human alpha S1-casein like protein: purification and N-terminal sequence determination.

Casein components of human milk are generally reported to belong to the beta- and kappa-groups. In this research human casein fraction was obtained from pooled mature milk, either by ultracentrifugation or by acid precipitation. In both cases a minor component with a slightly higher mobility in SDS-PAGE than beta-casein was identified. The protein was purified to homogeneity, the N-terminal sequence of the first 14 amino acid residues of this new human casein subunit shows a high degree of homology with the alpha s1-casein sequences from other species.

Modulation of Na+/K+ pump in intact erythrocytes by cardioglycosides, steroid hormones and ouabain-like compounds.

1. Pure erythrocytes preparations, free from platelets and white cells, were incubated for a long time without hemolysis. 2. Dose-response experiments performed with (a) cardioglycosides (ouabain and K-strophantoside), (b) steroid hormones and their glucuronides (tetrahydrocortisol, oestradiol and the respective 3-glucuronic derivatives) and (c) ouabain-like compounds purified in our laboratory (0.7 kDa and 2-4 kDa respectively) emphasise a modulatory effect [activation of Na+ efflux rate and K+ uptake at very low ligand concentrations, inhibition at higher levels; maximum enhancement of cation transport: (a) and (b) 10-0.1 nM (+40-50%), (c) 1-0.01 nM (2.5-fold)]. 3. Binding experiments show upward-curved Scatchard graphs, with the Kd values of 50 nM and 18 microM and the Bmax values of 10.2 and 984.5 fmol/100 microliters RBC (red blood cells) respectively.

Modulatory effect of two cardioglycosides on reconstituted Na+/K(+)-ATPase in proteoliposomes.

1. Na,K-ATPase was extracted from Cavia cobaya kidneys, solubilized with nonionic detergent C12E8 (octaethyleneglycol dodecyl monoether) in mixed lipid-detergent-protein micelles. The Na,K-ATPase specific activity was 30-35 IU/mg protein. 2. The enzyme was reconstituted in vesicles, made of phosphatidylethanolamine and cholesterol: an enhancement of +60% in specific activity was obtained. 3. Two different vesicle-types were carried out: open liposomes (partially organized membranes) and closed liposomes. 4. Proteoliposomes were employed for measuring the modulatory effect of two cardioglycosides: ouabain and digoxin. 5. Inhibition of the Na,K-ATPase activity revealed apparent Ki of 1.25 microM for ouabain and 0.25 microM for digoxin in open liposomes, and apparent Ki of 0.75 microM for ouabain and of 1.75 microM for digoxin in closed liposomes. 6. Maximum enhancement of enzymatic activity was found at concentrations of 5-0.5 nM for ouabain and 5-1 nM for digoxin in open liposomes, and 25-1 nM for both digoxin and ouabain in closed liposomes.

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus.

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.

Modulatory effect of some steroid hormones, their glucuronides and ouabain-like compounds on Cavia cobaya kidney Na+,K(+)-ATPase activity.

1. Ouabain-like compounds (approx. mol. wt 700, 2,000 and 4,000 Da) were purified from plasma of essential hypertensive patients. 2. Dose-response experiments performed with (a) steroid hormones, (b) their glucuronides and (c) ouabain-like compounds, emphasize a modulatory effect [activation of the Na,K-ATPase at very low concentrations of ligand, inhibition at higher levels; apparent Ki: (a) between 1 and 0.5 mM; (b) between 1 and 0.5 microM; and (c) between 10 and 1 nM; maximum enhancement of the enzymatic activity: (a) +20%; (b) +45%; and (c) +100%]. 3. Displacement experiments of [3H]ouabain evidence a high competition of the ligands towards the cardioglycoside. The relative I50s are: (a) between 1 and 0.5 mM; (b) between 10 and 1 microM; and (c) between 10 and 0.01 nM.