Ala214,38]aprotinin: preparation by partial desulphurization of aprotinin by means of Raney nickel and comparison with other aprotinin derivatives

Treatment of aprotinin with Raney nickel in the presence or absence of denaturants yielded [Ala2 14,38]aprotinin. Aprotinin and [Ala2 14,38]aprotinin were separated by ion exchange chromatography at pH 8 using CM-Sepharose, fast flow. [Ala2 14,38]aprotinin is a proteinase inhibitor, but it possesses lower affinities than aprotinin, for the enzymes trypsin, alpha-chymotrypsin, pancreatic kallikrein and plasmin as reflected by higher Ki values [Ala2 14,38]aprotinin is slowly degraded by trypsin. The optical activity of [Ala2 14,38]aprotinin in different solvents is quite similar to that of aprotinin, or that of its hydrolysis products, [seco-15/16]aprotinin or [di-seco-15/16,39/40]-aprotinin. This is taken as good evidence for analogous molecular conformations of all these substances.     

Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz).

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.     

Effect of covalent binding of aprotinin with a polysaccharide carrier on its inhibition of kallikrein from human plasma, porcine pancreatic kallikrein and trypsin

The inhibition of trypsin, human blood plasma kallikrein and porcine pancreatic kallikrein by aprotinin (native and immobilized on carboxymethyl ester of dextran) was investigated. The experimental values of Ki of native and immobilized aprotinin--enzyme complexes are equal to 0.037 and 0.045 nM for trypsin, 0.38 and 112.3 nM for pancreatic kallikrein and 34.4 and 454.5 nM for plasma kallikrein with N alpha-benzoyl-L-arginine ethyl ester as substrate, and to 82.6 and 231.7 nM for plasma kallikrein with a natural substrate--kininogen. These data suggest that covalent binding of aprotinin to the water-soluble polysaccharide carrier does not interfere with its interaction with trypsin, whereas the inhibition of kallikreins decreases, especially that of pancreatic kallikrein. The experimental results indicate the marked differences in the structure of the binding site of the active center (or its environment) of plasma and pancreatic kallikreins, on one hand, and trypsin, on the other, as well as the differences between the plasma and pancreatic kallikreins. A high requirement of kallikreins to the maintenance of the native conformation of aprotinin during immobilization is postulated.     

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.     

ThepH dependence of the equilibrium constantK Hyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond in bovine pancreatic trypsin inhibitor (aprotinin)

ThepH dependence of the equilibrium constant K_Hyd for the hydrolysis of the Lys¹⁵-Ala¹⁶ reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over thepH range 2.3–6.5. Solutions of aprotinin, modified aprotinin with the Lys¹⁵-Ala¹⁶ peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine β-trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The K_Hyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. ThepH-independent equilibrium constant is 0.90 and thepK values of the Lys¹⁵ carboxyl group and of the Ala¹⁶ amino group are 3.10 and 8.22, respectively. ThepK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.     

Chemical semisynthesis of aprotinin homologues and derivatives mutated inp′ positions

An extended concept for the replacement of amino acids in theP' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys¹⁵-carboxyl group) lacking the amino acids Ala¹⁶ and Arg¹⁷ leads to aprotinin homologues and derivatives mutated in theP′ ₁ andP′ ₂ position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala¹⁷]BPTI and [seco-17/18]BPTI is described in detail.     

The pH dependence of the equilibrium constant KHyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond in bovine pancreatic trypsin inhibitor (aprotinin)

ThepH dependence of the equilibrium constant K_Hyd for the hydrolysis of the Lys¹⁵-Ala¹⁶ reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over thepH range 2.3–6.5. Solutions of aprotinin, modified aprotinin with the Lys¹⁵-Ala¹⁶ peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine -trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The K_Hyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. ThepH-independent equilibrium constant is 0.90 and thepK values of the Lys¹⁵ carboxyl group and of the Ala¹⁶ amino group are 3.10 and 8.22, respectively. ThepK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.     

Structural characterization by nuclear magnetic resonance of a reactive-site 13carbon-labelled basic pancreatic trypsin inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed

With the use of an enzymatic replacement method, 90%-enriched [1-13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive-site peptide bond Lys-15--Ala-16 was properly resynthesized, the polypeptide chain was cleaved at the peptide bond Arg-39--Ala-40 and Arg-39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi-state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the [1-13C]lys-15-labelled inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase-inhibitor interactions.     

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

Conformation and inhibitory properties of peptides based on the tissue kallikrein-aprotinin complex.

A series of peptides encompassing the primary binding segment (residues 12-19) of aprotinin has been synthesized and tested for their ability to inhibit porcine pancreatic kallikrein. A minimum sequence of five amino acids spanning residues 12-16 of aprotinin is necessary for inhibition of porcine pancreatic kallikrein. An octapeptide homologous with the binding segment of aprotinin has a Ki-value of 1.2 x 10(-4) M. The solution structure of the octapeptide was studied by one- and two-dimensional NMR methods for comparison with the known structure of the segment of aprotinin that contacts tissue kallikrein. NMR experiments suggest that the peptide is either a random coil or that it samples several conformations on the NMR time scale. Analysis of the molecular dynamics trajectory of the octapeptide also suggests that the peptide is highly flexible. Thus, inhibition by the octapeptide occurs because of its homology with residues 12-19 of aprotinin. Moreover, the absence of a stable solution conformation similar to that of the binding segment of aprotinin is consistent with the 150,000-fold increase in Ki of the octapeptide compared to intact aprotinin.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor: Crystallization,patterson search,structure determination,refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibitor complex

The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P4₁2₁2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.The root-mean-square deviation, including all main-chain atoms, is 0·45 Å and 0·65 Å for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 Å away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 Å hydrogen bond with His57 NE.The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al., 1983).     

Chemical semisynthesis of aprotinin homologues and derivatives mutated inp′ positions

An extended concept for the replacement of amino acids in theP' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys¹⁵-carboxyl group) lacking the amino acids Ala¹⁶ and Arg¹⁷ leads to aprotinin homologues and derivatives mutated in theP ₁ andP ₂ position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala¹⁷]BPTI and [seco-17/18]BPTI is described in detail.     

Identification, purification, and localization of tissue kallikrein in rat heart.

A tissue kallikrein has been isolated from rat heart extracts by DEAE-Sepharose and aprotinin-affinity column chromatography. The purified cardiac enzyme has both N-tosyl-L-arginine methyl ester esterolytic and kinin-releasing activities, and displays parallelism with standard curves in a kallikrein radioimmunoassay, indicating it to have immunological identity with tissue kallikrein. The enzyme is inhibited by aprotinin, antipain, leupeptin and by high concentrations of soybean trypsin inhibitor, but stimulated by lima-bean or ovomucoid trypsin inhibitor and low concentrations of soybean trypsin inhibitor. By using a specific monoclonal antibody to tissue kallikrein in Western blot as well as active-site labelling with [14C]di-isopropyl fluorophosphate, the cardiac enzyme was identified as a protein of 38 kDa, a molecular mass identical with that of tissue kallikrein. Immunocytochemistry at the electron-microscopic level localized this enzyme to the sarcoplasmic reticulum and granules of rat atrial myocytes. Two cardiac kallikrein precursors, (38 and 40 kDa) were identified from the translation in vitro of heart mRNA by immunoprecipitation and electrophoresis of [35S]methionine-labelled cell-free translation products. Kallikrein mRNA in the rat heart was also demonstrated by dot-blot analysis using a tissue kallikrein cDNA probe. These results indicate that the tissue kallikrein gene is expressed in the rat heart and that the purified enzyme is indistinguishable from tissue kallikrein with respect to enzymic and immunological characteristics.     

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.     

Identification of [hydroxyproline3]-bradykinin released from human plasma and plasma protein Cohn's fraction IV-4 by trypsin

Aside from bradykinin (BK), a novel kinin, [Hydroxyproline3]-bradykinin ( [Hyp3]-BK), was isolated from the reaction mixture of human plasma and plasma protein Cohn's fraction IV-4 with trypsin. The liberated kinins were isolated based on procedures which we previously described for the isolation of [Hyp3]-lysyl-bradykinin ( [Hyp3]-Lys-BK) formed by kallikrein. The ratio of the amounts of two kinins thus formed from human plasma protein Cohn's fraction IV-4 were [Hyp3]-BK 25 +/- 4% and BK 75 +/- 4%, similarly to that of [Hyp3]-Lys-BK and Lys-BK, formed by kallikrein, but it varied by persons. The isolation of [Hyp3]-BK and [Hyp3]-Lys-BK suggests that a novel kininogen containing hydroxyproline in the third position of the bradykinin sequence in human plasma protein, possibly undergone post-translational modifications.     

Synthesis, cloning and expression of recombinant aprotinin

Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E. coli. The synthetic gene is characterized by the presence of certain restriction sites. These restriction sites are unique within the used cloning system. Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments. Using this method the variant [Glu52]aprotinin was obtained starting from the aprotinin gene. Both genes were successfully expressed in E. coli as fusion proteins with beta-galactosidase using vector pUR 278. No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8). [Glu52]aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein. This recombinant [Glu52]aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin.     

Preparation of chemically 'mutated' aprotinin homologues by semisynthesis. P1 substitutions change inhibitory specificity

The semisynthesis of homologues of aprotinin (BPTI) is described. The P1 amino acid residue of these homologues was substituted by other amino acids using peptide synthetic methods. The reactive-site-modified inhibitor (with the Lys15-Ala16 peptide bond hydrolyzed) was used as starting material. All carboxyl groups of the modified inhibitor were esterified with methanol, then the Lys15 methyl ester group was hydrolyzed selectively. Afterwards, Lys15 itself was split off. A new amino acid residue was incorporated by using water-soluble carbodiimide combined with an acylation catalyst. tert-Butyl-ester-protected amino acids were used for reinsertion. The method was tested by re-insertion of Lys15 to reconstitute the original inhibitor. Thirteen BPTI homologues with coded (Lys, Glu, Gly, Ala, Val, Ile, Leu) or uncoded amino acids (Abu, Ape, aIle, Ahx, tLeu, Neo) in position 15 were synthesized and the specificity of the inhibitors investigated. Amongst these, [Val15]BPTI was shown to be an excellent inhibitor for human polymorphonuclear leukocyte elastase having a complex dissociation constant of 0.11 nM. This inhibitor showed no detectable affinity to bovine pancreatic trypsin.     

Comparison of Tryptic Peptides of Benzodiazepine Binding Proteins Photolabeled with [3H]Flunitrazepam or [3H]Ro 15–4513

When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15-4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15-4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]flunitrazepam or [3H]Ro 15-4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.