The venom of the black mamba contains a

The screening of a bovine submaxillary gland cDNA library yielded 25 clones coding for bovine lactotransferrin. The nucleotide sequence of the longest insert contained a protein-coding region of 21 15 nucleotides and a 3' non-coding region of 194 nucleotides followed by a poly(A) tract of about 55 nucleotides. The predicted peptide sequence included a 16-amino-acid signal sequence upstream of the first amino acid of the native protein.The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the N-terminal and tryptic peptide sequences derived from purified bovine milk lactotransferrin, and also by similarity with human and murine lactotransferrins. The cDNA described corresponds to a 705-amino-acid-long preprotein that lacks the start methionine. The sequence of the secreted protein is 689 amino acids long and contains five potential glycosylation sites. Bovine lactotransferrin is 69% and 64% identical to human and murine lactotransferrins, respectively.The transferrins are a family of non-haem, iron-binding proteins which includes serum transferrin, lactotransferrin and melanotransferrin. They are monomeric glycoproteins with a molecular mass of about 80 kDa constituted by two lobes, each possessing one iron-binding site with the capacity to bind reversibly one ferric ion (Fe3+) (reviewed in [l, 21). Lactotransferrins (also called lactoferrins) are present in various biological fluids such as human milk [3 -51, bovine milk [6], saliva [7, 81 and mucous secretions [8, 91. They are also present in leucocytes [S, 10, 111. Although lactotransferrins were first isolated in 1960 from both human and bovine milk, most of the studies concerning their structure and biological roles (reviewed in [l, 21 and [12 -141) were performed on the human protein. The sequence of human lactotransferrin was resolved using chemical methods [15] and cDNA analysis [16] (and cited by Anderson et al. in [17]). It consists of a 691-amino-acid polypeptide chain to which two biantennary glycans of the N-acetyllactosaminic type are linked [18]. This sequence is 70% identical to the sequence of murine lactotransferrin recently determined by cDNA sequencing [19] and resolved as a 688-amino-acid polypeptide chain to which a single glycan of the N-acetyllactosaminic type is conjugated 1201.The only information known about the primary structure of bovine lactotransferrin concerned its N-terminal amino acid sequence APRKNVRWXTISQPE [21] and its carboCorrespondence to A.

Nacystelyn (NAL), a recently-developed lysine salt of N-acetylcysteine (NAC), and NAG, both known to have excellent mucolytic capabilities, were tested for their ability to enhance cellular antioxidant defence mechanisms. To accomplish this, both drugs were tested in vitro for their capacity: (1) to inhibit O2- and H2O2 in cell-free assay systems; (2) to reduce O2- and H2O2 released by polymorphonuclear leukocytes (PMN); and (3) for their cellular glutathione (GSH) precursor effect. In comparison with GSH, NAL and NAC inhibited H2O2, but not O2-, in cell-free, in vitro test systems in a similar manner. The anti-H2O2 effect of these drugs was as potent as that of GSH, an important antioxidant in mammalian cells. To enhance cellular GSH levels, increasing concentrations (0-2 x 10(-4) mol l-1) of both substances were added to a transformed alveolar cell line (A549 cells). After NAC administration (2 x 10(-4) mol l-1), total intracellular GSH (GSH + 2GSSG) levels reached 4.5 +/- 1.1 x 10(-6) mol per 10(6) cells, whereas NAL increased GSH to 8.3 +/- 1.6 x 10(-6) mol per 10(6) cells. NAC and NAL administration also induced extracellular GSH secretion; about two-fold (NAC), and 1.5-fold (NAL), respectively. The GSH precursor potency of cystine was about two-fold higher than that of NAL and NAC, indicating that the deacetylation process of NAL and NAC slows the ability of both drugs to induce cellular glut production and secretion. Buthionine-sulphoximine, which is an inhibitor of GSH synthetase, blocked the cellular GSH precursor effect of all substances. In addition, these data demonstrate that NAC and NAL reduce H2O2 released by freshly-isolated cultured blood PMN from smokers with chronic obstructive pulmonary disease (COPD) (n = 10) in a similar manner (about 45% reduction of H2O2 activity by NAC or NAL at 4 x 10(-6) mol l-1). In accordance with the results obtained from cell-free, in vitro assays, O2- released by PMN was not affected. Ambroxol (concentrations: 10(-9)-10(-3) mol l-1) did not reduce activity levels of H2O2 and O2- in vitro. Due to the basic effect of dissolved lysine, which separates easily in solution from NAL, the acidic function of the remaining NAC molecule is almost completely neutralized [at concentration 2 x 10(-4) M: pH 3.6 (NAC), pH 6.4 (NAL)]. Due to their function as H2O2 scavengers, and due to their ability to enhance cellular glutathione levels, NAL and NAC both have potent antioxidant capabilities in vitro. The advantage of NAL over NAC is two-fold; it enhances intracellular GSH levels twice as effectively, and it forms neutral pH solutions whereas NAC is acidic. Concluding from these in vitro results, NAL could be an interesting alternative to enhance the antioxidant capacity at the epithelial surface of the lung by aerosol administration.

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na' channel. It has been used to purify the porcine kidney amiloride-binding protein. analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (it) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

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Inter-a-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as HI and H 2 , respectively.We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H 3 inside pre-a-trypsin inhibitor (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type ./V-glycans in both H! and H 2 and that of O-glycans in H 2 . HI is eluted from Con-A Sepharose by cc-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H 2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates.The capacity of H 2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI. Die schweren Ketten des Inter-a-Trypsininhibitors: Isolierung und Identifizierung durch Elektrophorese und Teilsequenzierung. Unterschiedliche Reaktivitäten gegenüber Concanavalin A.Zusammenfassung: Der Inter-a-Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten Bikunin besteht sowie aus zwei schweren Ketten mit den apparenten M r -Werten 96000 und 86000 (in der SDS/PAGE unter nicht reduzierenden Bedingungen). Durch Sequenzanalyse konnten wir diese beiden Komponenten klar als HI und H 2 identifizieren.Wir zeigen, daß sowohl alkalische Behandlung (50mM NaOH 5 min bei Raumtemperatur) wie Chondroitase-Verdauung zur Dissoziation von ITI führen. Die für die Alkalibehandlung verwendeten Bedingungen wurden bereits für die Spaltung der kovalenten Bindung zwischen Bikunin und H 3 im Prä-atrypsininhibitor mitgeteilt (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).

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