A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 lU/mg and dissociation constants K { of the thrombin-inhibitor complexes close to 3 10m ol//. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7 087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-0-sulfate residue near the C-terminus. Isolierung und Charakterisierung von Hirudin-Isoinhibitoren und Sequenzanalyse von Hirudin PAZusammenfassung: Zur Isolierung von Isoformen des Hirudins (Isohirudinen) aus Blutegeln wurde eine Reinigungsprozedur entwickelt, die fünf Stufen umfaßt. Die dabei erhaltenen zwei Hemmstoff-Fraktionen wurden mittels HPLC weiter aufgetrennt, wobei mehrere Isohirudine erhalten wurden, die N-terminal entweder einen Valin-oder Isoleucinrest besitzen. Die Isohirudine sind gegenüber Thrombin vergleichbar hemmaktiv; die Dissoziations-Konstanten der Komplexe liegen bei 3 l O"" 11 mol//. Der Isoinhibitor mit N-terminalem Isoleucin, Hirudin PA, hat 66 Aminosäurereste und eine Molekülmasse von 7 087 Da. Mittels automatischem Edmanabbau des nativen und oxidierten Inhibitors und zweier tryptischer Fragmente konnte die komplette Aminosäuresequenz von Hirudin PA aufgeklärt werden. Auf der Basis der Primärstrukturen lassen sich nun zwei Isohirudine unterscheiden, das bereits länger bekannte Hirudin und das hier erstmals beschriebene Hirudin PA. Die strukturelle Homologie der Isoformen liegt bei 82% und sie enthalten beide einen Tyrosin-O-sulfat-Rest nahe dem C-Terminus.

Hirudin, the thrpmbin-specific inhibitor from the leech Hirudo medicinalis, is a single-chain polypeptide (65 amino-acid residues) linked by three disulfide bridges. Localization of the three disulfide bonds could be assigned on the basis of the structures of cystine peptides derived by high performance liquid chromatography separations of thermolysinolytic digest of native hirudin. By characterization of the nine major fragments by aminoacid analysis, N-terminal amino-acid determination and sequence analysis, the following disulfide linkages were identified: Cys 6 -Cys 14 , Cys 16 -Cys 28 and Cys 22 -Cys 39 . Due to the lack of any closer sequence homology and topological structural homology to other serine proteinase inhibitor proteins, hirudin seems to be unique in its primary structure and hence designates an unknown inhibitor family.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.

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Specific anti-30S protein immunoglobulin G fragments (Fab) were used to determine the contribution of each of the 30S ribosomal proteins to: (1) polyphenylalanine synthesis, (2) initiation factor-dependent binding of fMet-tRNA, (3) T-factor-dependent binding of phenylalanyl-tRNA, and (4) fixation of radioactive dihydrostreptomycin. Twenty of the 21 possible antibodies (antibody against S17 excepted) were used. In conditions where all the 30S proteins were accessible to Fabs, all of these monovalent antibodies strongly inhibited polyphenylalanine synthesis in vitro. Antibodies against S4, S6, S7, S12, S15, and S16, however, showed a weaker effect.30S proteins can be classified into four categories by their contributions to the function of sites "A" and "P":class I appears nonessential for tRNA positioning at either site (S4, S7, S15, and S16); class II includes proteins whose role in initiation is critical (S2, S5, S6, S12, and S13); class III (S8, S9, Sli, and S18) corresponds to proteins whose blockade prevents internal (elongation factor Tudependent) positioning; and class IV includes entities that are essential for activities of both "A" and "P" sites (SI, S3, S1O, S14, S19, S20, and S21). Dihydrostreptomycin fixation to the 30S or 70S ribosomes was inhibited by antibodies against S1, S1O, Sli, S18, S19, S20, and S21, but only weakly by the anti-S12 (Str A protein) Fab;The significance of these results is discussed in relation to 30S protein function, heterogeneity, and topography.Attempts have been made to identify the individual functions in protein synthesis of some of the ribosomal proteins. Reconstitution of 308 subunits with mixtures of protein lacking individual components (1) or addition of individual components to intact 30S subunits (2) have provided preliminary functional data for some of the 30S proteins. We describe another approach to this problem. Fab immunoglobulin fragments derived from specific antisera raised against individual ribosomal proteins (3-5) are used to inactivate 30S subunit function in several specific steps of protein synthesis. In particular, our data permit a tentative identification of those 30S proteins that constitute or are close to the sites for initiation factor (IF)-fMet-tRNA or elongation factor Tu (EF-Tu)-Phe-tRNA complexes and streptomycin fixation. We find that there are a relatively large number of proteins involved in these functions. Some appear to play a specific role while others have common functions in the various steps. MATERIALS AND METHODSImmunoglobulins were prepared from specific antisera by precipitation with (NH4)2SO4. The redissolved precipitate was applied to a Sephadex G-150 column to separate the 7S immunoglobulin Gs. In some preparations, a further purification by DEAE-cellulose column chromatography was performed (5). Fab was prepared as described (6). Only the Fab I fraction was used. A more detailed description of these methods is given elsewhere (5). The precipitation titers (3).of the immunoglobulin Gs used for different Fab prepara...