Binding proteins for cyclic and linear oligopeptides in plasma membranes and the cytosol of rat hepatocytes.

Using a cyclolinopeptide A analogue, the hydrophobic cyclic peptide c(-Ala-Lys-Pro-Phe-Phe-Ala-Lys-Pro-Phe-Phe-), termed CDP (cyclodecapeptide), as ligand in affinity chromatography, hepatocellular peptide binding proteins were isolated from the integral part of plasma membranes and the cytosol. The sequence of the isolated protein with MW of 50 kDa from the integral part of the plasma membrane fraction was identical to cytochrome P450 II C13 and cytochrome P450 II C22, whereas the sequence of the 54 kDa protein was identical to 3-hydroxyandrogen-UDP-glucuronosyltransferase. These proteins have also been described as binding proteins for bile acids. As shown in earlier studies, bile acids and CDP also compete for uptake into hepatocytes. In the cytosol, a further known bile acid binding protein, the glutathione-S-transferase (G-S-T) subunit Yb1, was isolated and sequenced as binding protein for CDP and also for a further cyclopeptide, the somatostatin analogue OO8, and a linear peptide with renin-inhibiting activity, EMD 55068. As shown in uptake studies using isolated basolateral plasma membrane vesicles, G-S-T was able to increase the uptake of EMD 51921, a linear peptide with renin-inhibiting potency, into the vesicles when the latter were preloaded with G-S-T. The binding of the substrate to the outside of the preloaded vesicles was not different than binding to unloaded vesicles. The maximal transport rate of the carrier-mediated/facilitated diffusion and the rate of permeation, however, were doubled in the presence of G-S-T, pointing to the involvement of intracellular binding proteins such as G-S-T in the unloading of the carrier protein and in the reduction of the free substrate concentration.

A fraction enriched in a novel glucocorticoid receptor-interacting protein stimulates receptor-dependent transcription in vitro.

Glucocorticoids influence numerous cell functions by regulating gene activity. The glucocorticoid receptor (GR) is a ligand-activated transcription factor and, like any other transcription factor, does not modulate gene activity just by binding to DNA. Interaction with other proteins is probably required to enhance the establishment of a functional transcription initiation complex. To identify such proteins, we analyzed the in vitro interaction of the glucocorticoid receptor bound to a double glucocorticoid response element with nuclear proteins and describe here three interacting proteins with different molecular weights. One of them, which we named GRIP 170 (GR-interacting protein), was purified and microsequenced, and it turned out to be an unknown protein. When tested in a cell-free transcription assay, the fraction highly enriched for GRIP 170 does not influence basal promoter activity but does enhance GR induction.

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

The 60-kDa bumetanide-binding protein from rat liver membranes is a catalase.

A hepatic bumetanide-binding protein of molecular mass 60 kDa was isolated from rat liver sinusoidal plasma membranes after photoaffinity labelling with [3H]bumetanide. The protein was purified by non-equilibrium pH gel electrophoresis/two-dimensional gel electrophoresis. The amino acid sequences of two internal fragments share 67% and 89% similarity with rat liver catalase, which has a molecular mass of 59.758 kDa. With H2O2 as a substrate, the catalytic activity was measured in rat liver plasma membrane preparations. This activity was blocked by bumetanide and aminobumetanide. Polyclonal antibodies were raised against the purified 60-kDa membrane bumetanide-binding protein. The antibody anti-Bum-Ab 60 immunoprecipitated a 60-kDa protein from rat hepatocytes. Immunoblot analysis of SDS/PAGE and two-dimensional PAGE gels confirmed that the antibody was specific for the 60-kDa bumetanide-binding protein and cross-reacted with commercially available purified bovine liver catalase. Immunofluorescence showed the presence of the 60-kDa antigen in the plasma membrane of intact hepatocytes. Western-blot analysis revealed that the protein was present in rat kidney cortex homogenate but was lacking in hepatoma cells AS-30 D, Reuber H35 FAO and HPCT cells (clone 1E3), in spleen, and in ileum. These results indicate that a plasma-membrane-derived catalase binds bumetanide in rat liver.

Cyclic somatostatin analogs bind specifically to pI 6.1 carboxylesterase of rat liver cells.

The hydrophobic cyclohexapeptide cyclo(Phe-Thr-Lys-Trp-Phe-DPro) (008), an analog of somatostatin with retro sequence, was previously shown to competitively inhibit the uptake of cholate and taurocholate into isolated rat liver cells. Conversely, the competitive uptake inhibition of 008 into isolated rat hepatocytes by bile acids confirmed the observation of common binding and transport sites by bile acids and cyclosomatostatin. Furthermore the transport characteristics of 008 uptake revealed a significant and rapid binding to cell membranes. In this context it was of special interest to investigate the specificity of the binding component since specific binding of the substrate to membrane proteins could be responsible for the low Km of 008-transport. Therefore, the cyclohexapeptide 008 could be used as the ligand in affinity chromatography in order to isolate such binding proteins. The gel matrix used did not interact non-specifically with octylglucoside-solubilized proteins from isolated rat liver plasma membranes. In affinity chromatography of octylglucoside-solubilized plasma membranes, two dominant proteins with apparent molecular masses of 60 and 58 kDa bound specifically to the 008 ligand. When used as ligands in affinity chromatography, these membrane-associated 60 and 58 kDa proteins bound exclusively to aromatic cyclopeptides, e.g. cyclosomatostatin 008, but not to linear peptides or taurocholate derivatives. The amino acid sequences of tryptic digests of the 008-affinity-purified 58 kDa protein were identical to the sequence of a microsomal pI6.1 carboxylesterase. Immunofluorescence of intact hepatocytes showed that this xenobiotic metabolizing enzyme is also located in sinusoidal rat liver plasma membranes and could therefore account for the extensive and specific binding of the cyclosomatostatin to sinusoidal plasma membranes of rat liver.

Intestinal bile acid absorption. Na(+)-dependent bile acid transport activity in rabbit small intestine correlates with the coexpression of an integral 93-kDa and a peripheral 14-kDa bile acid-binding membrane protein along the duodenum-ileum axis.

The anatomical localization of the Na+/bile acid cotransport system from rabbit small intestine was determined using brush border membrane vesicles prepared from eight different segments of the small intestine. Na(+)-dependent transport activity for bile acids, both for [3H]taurocholate and [3H]cholate, was found in the distal segment 8 only representing the terminal 12% of the small intestine. In contrast, the Na(+)-dependent D-glucose transporter and the H(+)-dependent oligopeptide transporter were found over the whole length of rabbit small intestine in all segments. Photoaffinity labeling with 7,7-azo- and 3,3-azo-derivatives of taurocholate with subsequent fluorographic detection of labeled polypeptides after one- and two-dimensional gel electrophoresis showed that an integral membrane polypeptide of M(r) 87,000 is present in the entire small intestine, whereas an integral membrane protein of M(r) 93,000 together with a peripheral membrane protein of M(r) 14,000 are exclusively expressed in the distal small intestine correlating with Na(+)-dependent bile acid transport activity. Photoaffinity labeling with the cationic bile acid derivative 1-(7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta[3 beta-3H]cholan-24-oyl)-1,2- diaminoethane hydrochloride and 7,7-azo-3 alpha,12 beta-dihydroxy-5 beta[12 alpha-3H]cholan-24-oic acid did not result in a specific labeling of the above mentioned proteins, demonstrating their specificity for physiological bile acids. Photoaffinity labeling of the 93- and 14-kDa bile acid-binding proteins was strongly Na(+)-dependent. Significant labeling of the 93- and 14-kDa proteins occurred only in the presence of Na+ ions with maximal labeling above 100 mM [Na+] showing a parallel [Na+] dependence to transport activity. Inactivation of Na(+)-dependent [3H]taurocholate uptake by treatment of ileal brush border membrane vesicles with 4-nitrobenzo-2-oxa-1,3-diazol chloride led to a parallel decrease in the extent of photoaffinity labeling of both the 93- and 14-kDa protein. Sequence analysis of the membrane-bound 14-kDa bile acid-binding protein surprisingly revealed its identity with gastrotropin, a hydrophobic ligand-binding protein exclusively found in the cytosol from ileocytes and thought to be involved in the intracellular transport of bile acids from the brush border membrane to the basolateral pole of the ileocyte. In conclusion, the present studies suggest that both an integral 93- and a peripheral 14-kDa membrane protein, identified as gastrotropin, and both exclusively expressed in the terminal ileum, are essential components of the Na+/bile acid cotransport system in rabbit terminal ileum.

Charge heterogeneity of insulin fusion proteins expressed in Escherichia coli is not due to proteolytic degradation.

Evaluation of the yield of expression of exogeneous protein in transformed Escherichia coli cells by means of one-dimensional SDS-PAGE often leads to overestimation and miscalculation. For example, it is possible that proteins of similar size comigrate and thus mask the overexpressed product band. Therefore, two-dimensional electrophoresis was used to analyze two types of recombinant fusion proteins, i.e., a beta-galactosidase insulin fusion protein and a interleukin II insulin fusion protein, directly after fermentation. We found that production scale expression products show charge and size heterogeneity. The heterogeneous protein spots were characterized by subsequent blotting onto Immobilon membrane and by N-terminal sequencing. Some of the separated spots were either N-terminally blocked or already degraded to some extent. The integrity of the actual product component of the fusion protein was examined with a C-terminus-specific antibody and by Western blot analysis of the 2D gels.

Identification of novel phosphorylation sites in murine A-type lamins.

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.

The quinone-reactive Ni/Fe-hydrogenase of Wolinella succinogenes.

The hydrogenase (Hyd) isolated from the cytoplasmic membrane of Wolinella succinogenes consists of three polypeptides (HydA, HydB and HydC) and contains cytochrome b (6.4 mumol/g protein), which was reduced upon the addition of H2. The enzyme catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone with H2, in contrast to an earlier preparation which was made up of HydA and HydB only and did not contain cytochrome b (Unden, G., Böcher, R., Knecht, J. & Kröger, A. (1982) FEBS Lett. 145, 230-234). This suggests that HydC is a cytochrome b which serves as a mediator in the electron transfer from H2 to the quinone. The hydrogenase genes were cloned, sequenced and identified by sequence comparison with the N-termini of the three subunits. The three genes were arranged in the order hydA, hydB, hydC, with the transcription start site in front of hydA, and were present only once on the genome. Separated by an intergene region of 69 nucleotides, hydC was followed by at least two more open reading frames of unknown function. The amino acid sequences derived from hydA, hydB and hydC were similar to those of the membrane Ni-hydrogenases of seven other bacteria. HydA and HydB also showed similarity to the small and the large subunits of periplasmic Ni-hydrogenases. HydC was predicted to contain four hydrophobic segments which might span the bacterial membrane. Two histidine residues located in hydrophobic segments are conserved in the corresponding sequences of the other membrane hydrogenases and might ligate the haem B.

Isolation and characterization of an abundant elastase inhibitor from NaCl extracts of bovine nasal septa and articular cartilage.

Extracts of cartilage have been reported to inhibit many serine proteinases and metalloenzymes. Such inhibition may be important in protecting cartilage against degradation by chondrocytic proteinases such as collagenase, stromelysin and by leukocytic proteases, such as elastase. We report here isolation and partial characterization of a 17-kD elastase inhibitor from 0.5 M NaCl extracts of both nasal septum cartilage and articular cartilage, which inhibits elastase and represents 0.08% of the weight of nasal cartilage and 0.002% of the weight of articular cartilage. The protein was highly specific for elastase and did not inhibit cartilage metalloproteinases, suggesting that it may be mainly directed toward protecting cartilage against leukocytic proteases. The inhibitor had a blocked amino-terminus, was high in serine and glycine and lacked carbohydrate. The ease with which the inhibitor was extracted from cartilage suggests that it may function in vivo as a highly abundant elastase inhibitor which is secreted into synovial fluid from cartilage. The inhibitor was shown to be synthesized by bovine articular cartilage in explant culture and nearly all of the metabolically labeled material was secreted into the culture media. The inhibitor cross-reacted with polyclonal antibodies to bovine neck ligament alpha-elastin and antibodies to the inhibitor reacted with bovine neck ligament elastin. The properties of this inhibitor are different than those of any other reported cartilage derived inhibitor.

Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing.

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser390 and Ser392 and to PKC at Ser572. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2-kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.

Undulin is a novel member of the fibronectin-tenascin family of extracellular matrix glycoproteins.

We characterized cDNA clones specific for the extracellular matrix glycoprotein undulin. Two sets of cDNA clones were isolated from a human placental lambda gt11 expression library and from a rhabdomyosarcoma cell line encoding two partially identical carboxyl-terminal polypeptides of 843 (Un1) and 443 (Un2) amino acids suggesting differential splicing of a single gene transcript. Northern blot analysis of human rhabdomyosarcoma cell poly (A) RNA with cDNA specific for Un1 identified transcripts of approximately 4.2, 6.5, and 8.5 kilobases, whereas a probe specific for Un2 detected a single mRNA of approximately 5 kilobases. Since a monoclonal antibody that is reactive with a sequence encoded by Un1 and not by Un2 detects the bands considered characteristic for undulin in Western blots, the mRNAs related to Un1 may code for the major part of the undulin molecule. The protein sequences deduced from Un1 and Un2 reveal an amino-terminal differentially spliced von Willebrand factor A domain, characteristic of proteins that interact with interstitial collagens, which is linked to fibronectin-like type III homology units by a unique sequence of 57 amino acids. Whereas Un2 encodes two complete and one incomplete type III homologies followed by a unique acidic carboxyl-terminal domain of 118 amino acids, Un1 codes for seven complete and one truncated type III homologies, followed by a short proline-rich carboxyl-terminal segment of 23 amino acids. Considering the 298 amino acids occurring in identical segments, the 989 different amino acid positions deduced from clones Un1 and Un2 represent an estimated 40% of the overall undulin sequence. In the context of 1) rotary shadowing electron microscopy data showing undulin as a structure composed of nodules that are interconnected by flexible rods of varying size, 2) the presence of three major bands of Mr 270,000, 190,000, and 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 3) common antigenic epitopes and similar peptide maps (Schuppan, D., Cantaluppi, M.C., Becker, J., Veit, A., Bunte, T., Troyer, D., Schuppan, F., Schmid, M., Ackermann, R., and Hahn, E.G. (1990) J. Biol. Chem. 265, 8823-8832), our finding of differentially spliced type III homology units, as found in tenascin and fibronectin, suggests that undulin is another member of the fibronectin-tenascin family of extracellular matrix glycoproteins. Furthermore, as in fibronectin and tenascin, undulin bears an additional subset of interactive domains tailored to specific structural and functional roles in development and differentiation.

Disulphide bridge formation of proinsulin fusion proteins during secretion in Streptomyces.

To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and re-oxidation followed by proteolytic digestions led to insulins which are identical to authentic material. The ease of correct disulphide bond formation in solution and incorrect processing during secretion suggests involvement of yet unknown factors leading to an unfavourable folding of proinsulin.

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase.

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653-658) the total sequence is not known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713-722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme is characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.

Antithrombotic effects of recombinant hirudin in different animal models.

The effect of recombinant hirudin (r-hirudin; HBW 023) was studied in four different models of thrombosis as well as on bleeding time. Arteriolar thrombus formation was induced in rats either by argon laser injury or by photochemical reaction. r-Hirudin, given by intravenous infusion, significantly inhibited arteriolar thrombus formation and arterial bleeding time at a dose of 40 micrograms/kg.min. In rabbit jugular veins and carotid arteries, occluding thrombi were produced by stenosis and endothelial damage. r-Hirudin reduced the incidence of thrombosis dose dependently (ED50 0.7 and 1.0 mg/kg i.v. in arteries and veins, respectively). Caval vein thrombosis was initiated in rats by insertion of a stainless steel coil. This thrombosis was dose dependently reduced by the injection of r-hirudin (ED50 0.16 mg/kg i.v.). The duration of the antithrombotic effect of 0.25 mg/kg of r-hirudin in caval veins was about 60 min, whereas the arterial bleeding time was significantly prolonged in these rats for only 20 min. The plasma level of r-hirudin had dropped to 0.12 micrograms/ml 60 min after injection. These results suggest that the duration of the effect on bleeding time may be shorter than the antithrombotic action of r-hirudin.

Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b.

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli.

An aminotransferase capable of transaminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid to L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG), was purified to apparent homogeneity from Escherichia coli K-12. The enzyme catalyzes the transamination of L-phosphinothricin and various analogs with 2-ketoglutarate as the amino group acceptor. The transaminase has a molecular mass of 43 kilodaltons by sodium dodecyl sulfate-gel analysis and an isoelectric point of 4.35. The enzyme was most active in the high-pH region, with a maximum at pH 8.0 to 9.5, and had a temperature optimum of 55 degrees C. Heat stability was observed up to 70 degrees C. Substrate specificity studies suggested that the enzyme is identical with the 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19). The first 30 amino acids of the N terminus of the protein were determined by gas phase sequencing. The transaminase was immobilized by coupling to the epoxy-activated carrier VA-Biosynth (Riedel de Haen) and used in a column reactor for the continuous production of L-phosphinothricin. The enzyme reactor was operated for 7 weeks with only a slight loss of catalytic capacity. Production rates of more than 50 g of L-phosphinothricin per liter of column per h were obtained.

Primary structures of new 'iso-hirudins'.

Crude hirudin (12.7 U/micrograms), a complex mixture of polypeptides obtained from the leech, could be separated by microbore HPLC. A combination of amino acid analysis, N-terminal microsequencing and chemical as well as enzymatic fragmentation made the primary sequence of the new isohirudins Ia-IIIb' accessible. The biological activity determined in the thrombin inhibition test showed a comparable value for all of these compounds. The results presented address the question as to whether these isohirudins are true mutations from a family of genes or a family of leeches.