Expression, isolation and characterization of recombinant [Arg15,Glu52]aprotinin.

The [Arg15,Glu52]aprotinin gene has been constructed from a synthetic [Glu52]-aprotinin gene via an exchange of the appropriate DNA cassette. The gene has been fused to the N-terminal part of the bacteriophage MS-2 polymerase and expressed in a temperature inducible E. coli expression system. The produced fusion protein is deposited as inclusion bodies. Pure and functionally active [Arg15,Glu52]aprotinin has been obtained after cleavage of the purified fusion protein and renaturation of the aprotinin homologue. Recombinant [Arg15,Glu52]aprotinin shows good inhibition of human anionic and cationic trypsin (Ki less than or equal to 10(-11)M) and of human plasma kallikrein (Ki = 3.2 x 10(-10)M). The inhibition constants for human plasmin are Ki = 1.3 x 10(-10)M and for human urinary kallikrein Ki = 10(-11)M. No inhibition was found with the human proteinases thrombin, coagulation factor Xa, urokinase, tissue plasminogen activator, cathepsin G, leukocyte elastase and pancreatic elastase.

Complete amino acid sequence of the N-terminal extension of calf skin type III procollagen.

The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells.

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.

Inhibition of protein synthesis in vitro by procollagen-derived fragments is associated with changes in protein phosphorylation.

The NH2-terminal extension fragment (Col 1) of the pro alpha 1(I) procollagen chain selectively inhibits the translation of procollagen mRNA in a reticulocyte lysate system, whereas the reduced and alkylated fragment (AE-Col 1) and its proteolytically derived peptides inhibit the translation of all mRNAs (Hörlein, D., McPherson, J., Goh, S. H., and Bornstein, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6163-6167). The latter inhibitory function, which occurs at the level of polypeptide chain initiation, has now been shown to be associated with an increase in phosphorylation of an Mr = 94,000 protein. The time span required for observation of changes in phosphorylation and in inhibition of protein synthesis is similar. Since AE-Col 1 can serve as a substrate for casein kinase II, we suggest that phosphorylation of AE-Col 1 and its derivatives may be required for their activity.

Regulation of protein synthesis: translational control by procollagen-derived fragments.

Previous studies have shown that a type I procollagen-derived peptide, called pN alpha 1(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation of collagen synthesis, the physiological role of the lower molecular weight fragments, if any, is uncertain.

Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes.

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.

Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides.

Peptides prepared from the amino termini of pro alpha 1(I) and pro alpha 1(III) collagen chains inhibit the production of pro alpha 1(I) and pro alpha 2 by rat calvaria rna in a reticulocyte cell-free system. The synthesis of other proteins was not altered, suggesting a specific effect on collagen production. Various peptides from the helical region of the alpha 1(I) chain did not alter translation. These studies, taken together with earlier studies showing inhibition of collagen synthesis by cells in culture receiving the amino-terminal peptides, are consistent with a regulatory function in collagen synthesis for the amino-terminal peptides from procollagen.

Amino acid sequence of the aminoterminal segment of dermatosparactic calf-skin procollagen type I.

The N-terminal procollagen peptide of the pN alpha 1(I) chain from dermatosparactic calf skin contains 139 amino acid residues. For the determination of the amino acid sequence the procollagen peptide was treated with pyroglutamate aminopeptidase, protease from Staphylococcus aureus V8 and trypsin. The fragments obtained were separated by molecular sieve and ion-exchange chromatography and submitted to automated Edman degradation. The procollagen peptide consists of three segments, an N-terminal globular domain which contains all the cysteine residues and most of the hydrophobic residues present in the entire peptide, a triple helical part with a relatively high content of proline and hydroxyproline, and a short nonhelical region which forms the connection to the nonhelical region of the alpha 1(I) chain and which contains the proline-glutamine bond specifically split by the N-terminal procollagen peptidase during conversion of procollagen to collagen.

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.

Aminoterminal extension peptides from type I procollagen normalize excessive collagen synthesis of scleroderma fibroblasts.

Fibroblasts derived from a skin biopsy of a patient with scleroderma in the sclerotic stage were shown to have a higher rate of DNA synthesis, and to synthesize more collagen than fibroblasts from a healthy control. The addition of procollagen peptides to the culture medium of scleroderma fibroblasts almost normalized the collagen synthesis. This observation indicates that the mechanism for the regulation of collagen synthesis by feed back inhibition of prollagen peptides is functioning in this disease. It is suggested that the level of biologically active procollagen peptides is lowered.