A stoichiometric complex formed between human alpha‐thrombin and D‐Phe‐Pro‐Arg chloromethylketone was crystallized in an orthorhombic crystal form. Orientation and position of a starting model derived from homologous modelling were determined by Patterson search methods. The thrombin model was completed in a cyclic modelling‐crystallographic refinement procedure to a final R‐value of 0.171 for X‐ray data to 1.92 A. The structure is in full agreement with published cDNA sequence data. The A‐chain, ordered only in its central part, is positioned along the molecular surface opposite to the active site. The B‐chain exhibits the characteristic polypeptide fold of trypsin‐like proteinases. Several extended insertions form, however, large protuberances; most important for interaction with macromolecular substrates is the characteristic thrombin loop around Tyr60A‐Pro60B‐Pro60C‐Trp60D (chymotrypsinogen numbering) and the enlarged loop around the unique Trp148. The former considerably restricts the active site cleft and seems likely to be responsible for poor binding of most natural proteinase inhibitors to thrombin. The exceptional specificity of D‐Phe‐Pro‐Arg chloromethylketone can be explained by a hydrophobic cage formed by Ile174, Trp215, Leu99, His57, Tyr60A and Trp60D. The narrow active site cleft, with a more polar base and hydrophobic rims, extends towards the arginine‐rich surface of loop Lys70‐Glu80 that probably represents part of the anionic binding region for hirudin and fibrinogen.

The dissociation constant for hirudin was determined by varying the concentration of hirudin in the presence of a fixed concentration of thrombin and tripeptidyl p-nitroanilide substrate. The estimate of the dissociation constant determined in this manner displayed a dependence on the concentration of substrate which suggested the existence of two binding sites at which the substrate was able to compete with hirudin. A high-affinity site could be correlated with the binding of the substrate at the active site, and the other site had an affinity for the substrate that was 2 orders of magnitude lower. Extrapolation to zero substrate concentration yielded a value of 20 fM for the dissociation constant of hirudin at an ionic strength of 0.125. The dissociation constant for hirudin was markedly dependent on the ionic strength of the assay; it increased 20-fold when the ionic strength was increased from 0.1 to 0.4. This increase in dissociation constant was accompanied by a decrease in the rate with which hirudin associated with thrombin. This rate could be measured with a conventional recording spectrophotometer at higher ionic strength and was found to be independent of the binding of substrate at the active site.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha‐thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48‐65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35‐39 and 70‐80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N‐terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D‐Phe‐Pro‐ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate‐like inhibitor D‐Phe‐Pro‐ArgCH2Cl.

We report a new type of linkage between a carbohydrate and a protein, involving the rarely modified side chain of a tryptophan residue. An aldohexopyranosyl residue was found to be linked via a C-C bond to the indole ring of the tryptophan residue at position 7 of human RNase Us. Mass spectrometric analysis of peptides containing this residue showed a molecular mass 162 Da higher than that expected for tryptophan. The fragmentation pattern of the modified amino acid side chain was reminiscent of that of aromatic C-glycosides, suggesting a direct attachment of a hexose residue to a C-position of the tryptophan indole moiety. 'H and 13C NMR spectroscopic data confirmed this inference and unequivocally demonstrated the substituent to be an aldohexopyranosyl residue, C-glycosidically linked to the C2 atom of the indole. This mode of attachment differs from the ones known so far, in which carbohydrates are linked to an amino acid side chain by N-or 0-glycosidic bonds.

Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of ϳ60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an ␣-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C-and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.

Protein phosphatase 2A (polycation-stimulated protein phosphatase L) was purified from porcine kidney and skeletal muscle. The 36-kDa catalytic and the 65-kDa putative regulatory (hereafter termed PR65) subunits of protein phosphatase 2A2 were separated by reverse-phase HPLC. Partial amino acid sequence data (300 residues) was obtained for PR65. Molecular cloning showed that two distinct mRNAs (termed alpha and beta) encoded the PR65 subunit. The cDNA encoding the alpha-isotype spanned 2.2 kilobases (kb) and contained an open reading frame of 1767 bases predicting a protein of 65 kDa, which was in good agreement with the size of the purified protein. The cDNAs encoding the beta-isotype contained an open reading frame of size similar to that of alpha-form but lacked an initiator ATG. Northern analysis, using RNA isolated from several human cell lines, indicated that the alpha-isotype was encoded by a mRNA of 2.4 kb that was much more abundant than the beta mRNA of 4.0 kb. Comparison of the predicted amino acid sequences of the two isotypes revealed 87% identity. The deduced protein sequences of the alpha- and beta-isotypes were found to be made up of 15 imperfect repeating units consisting of 39 amino acids. This repeating structure was conserved between species.

cDNA clones coding for tenascin, an extracellular matrix glycoprotein with a restricted tissue distribution, were isolated from a chicken fibroblast cDNA expression library using a specific tenascin antiserum. Antibodies eluted from the cDNA‐encoded fusion proteins reacted exclusively with tenascin. Limited trypsin treatment of purified tenascin resulted in a peptide which confirmed the deduced protein sequences. The largest clone encoding 632 amino acids showed a cysteine‐rich region containing 13 consecutive epidermal growth factor‐like repeats of unusual uniformity. Northern blot analysis revealed 8‐ to 9‐kb messages. Tenascin is shown to be induced in vitro by fetal calf serum as well as by transforming growth factor beta (TGF‐beta). A 4‐fold increase in tenascin secretion by chick embryo fibroblasts was seen after TGF‐beta treatment. The induction of tenascin protein synthesis was preceded by an increase of tenascin mRNA as determined by Northern blot analysis. The induction of tenascin was compared with fibronectin. The accumulation of the two extracellular matrix proteins in the medium was differentially affected by fetal calf serum and TGF‐beta and the increase was in both cases higher for tenascin.

Tobacco glucan endo-1,3-fi-glucosidase (13-1,3-glucanase; 1,3-13-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues.,3-1,3-Glucanase is produced as a 359-residue preproenzyme