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The 6-kDa peptide of the human lactotransferrin N-terminal domain which inhibits binding of lactotransferrin to the phytohemagglutinin-activated lymphocyte was isolated. To examine the receptor-ligand cross-linking reaction, lactotransferrin was derivitized using two heterobifunctional reagents and was subsequently assayed to identify the derivitized lysine residues. In addition, molecular modeling was used characterize the position of the heterobifunctional reagents at protein surface. The results defined regions of human lactotransferrin that interacted with the lymphocyte receptor.

Human inter-α-inhibitor (IαI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan : a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IαI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-α-trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We mapped the disulphide topology of the IαI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IαI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IαI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated α-helicities of 24 % and 28 %, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment. Inside the IαI molecule, the heavy chains are linked to the glycosaminoglycan chain via... [ABSTRACT FROM AUTHOR]

Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the N-terminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) Eur. J. Biochem. 201,257– 2631, a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforrns (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with [14C]myristoyI-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC50, of about 0.1 μM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme. [ABSTRACT FROM AUTHOR]