Studies on brain opiate receptor stability in vitro: inhibition of opiate receptor binding by adenosine-5'-diphosphate

Incubation of rat brain homogenates at 37° causes a time-dependent decrease in opiate receptor binding which does not occur with a washed membrane fraction. The supernatant fraction contains a heat-stable inhibitor which is partially destroyed by apyrase and completely removed by activated charcoal. ADP causes a similar inhibitory effect in homogenates, but not with washed membranes, which is characterized by a decrease in both opiate agonist and antagonist binding in the absence or presence of NaCl. The ADP inhibition is antagonized by ATP, α,β-methyleneADP, β-thioADP and EDTA. It is concluded that ADP, unlike the guanine nucleotides, facilitates the nonspecific degradation of opiate receptors by an endogenous soluble factor.     

Characterization of bovine pancreatic ductal cells isolated by a perfusion-digestion technique

Summary Bovine pancreatic ductal cells isolated by perfusing an enzyme solution into the lumen of the main duct were obtained as sheets of cells. Morphologic features of these cells were those of pancreatic ductal epithelial cells. These cells also contained alcian blue/periodic acid-Schiff positive material and bound lectins, and they stained for keratin in the same manner as intact ductal epithelium. In culture, the plating efficiency was high (13.6%) as determined by DNA content before and after 24 h plating, perhaps due to the gentle isolation technique and the isolation of sheets of cells rather than a single cell. Cell doubling time was 34.4 h in Eagle's minimal essential medium with 10% heat inactivated fetal bovine serum and antibodies, and over 95% of the cell incorporated [³H]thymidine during a 6 h labeling period after 4 d in primary culture. Isolated cells grew best in medium CMRL 1066 with 10% heat inactivated fetal bovine serum as determined by measuring DNA content. The paper is Publication 1100 from the Department of Pathology, University of Maryland School of Medicine. This study was supported in part by National Cancer Institute (Bethesda, MD) Contract NO1-CP-75947 and Grant CA-19197-06 through the National Pancreatic Cancer Project.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

牛胰多肽对大鼠胰外分泌的抑制作用

在大鼠观察了牛胰多肽(BPP)对由蛋白胨和雨蛙素引起的大鼠胰外分泌的影响。向十二指肠灌入25%蛋白胨后90min 时,对照组的蛋白排出量约为其基础值的10倍,而1、3、5及10ug/kg BPP 剂量组的蛋白排出量分別为对照组的47%、33%、29%及29%。在灌注剂量为3μg/kg 雨蛙素后60min 时,对照组的蛋白排出量约为其基础值的14倍,但3、5及10μg/kgBPP 剂量组的蛋白排出量分別为对照组的74%、68%及55%。各组胰淀粉酶浓度的变化和其蛋白排出量变化相平行。胰液量的增加不受 BPP 影响。结果提示,BPP 能抑制胰酶的大量分泌,并有剂量依从关系,但不抑制胰液量。     

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.     

Electrostatic effect of trypsin binding on the hydrogen exchange rate of bovine pancreatic trypsin inhibitor beta-sheet NH's

The changes of H-D exchange rates upon protein-protein interactions are generally interpreted as a result of the changes of the dynamic properties of the proteins. The effect of trypsin binding on the H-D exchange kinetics of some trypsin inhibitor amide H's was reported (Simon et al., 1984). In this paper the electrostatic potential originating from the trypsin molecule is calculated at the positions of the studied amide H's in the trypsin-trypsin inhibitor complex. We conclude that the observed decrease of the exchange rates is mainly due to the electrostatic field of the trypsin molecule.     

Prediction of a low-frequency mode in bovine pancreatic trypsin inhibitor molecule

One of the frontiers today in molecular biology is to measure, identify and go further to predict the low-frequency internal motion of biological macromolecules, which is crucially important for understanding the dynamic mechanism of various biological functions occurring in such molecules. Based on the theory of continuity model developed recently for dealing with the internal low-frequency motion of a biological macromolecule, it is predicted that the low-frequency phonons with wave number of about 23 cm^?1 might be excited in BPTI molecule.     

Analysis of serine proteinase-inhibitor interaction by alanine shaving

We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving. One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s). Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor. We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability. To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis. Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)). In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects. However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds. Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction.     

Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state

Knowledge about the architecture of macromolecules has been derived primarily from crystallography. Therefore, it has been a matter of concern whether the conformation of a macromolecule in solution, namely in vivo, might be different from that in the crystalline state. To determine the difference between the conformations, a protein (trypsin inhibitor) dissolved in water has been simulated using the method of molecular dynamics and the results are compared with those obtained from a simulation of the full crystalline unit cell. We report here that no significant difference was found for backbone atoms, except for two more or less flexible loops extending from the core of the protein and the very flexible carboxyterminal residues. The side-chains in which the conformation in solution differs considerably from that in the crystal all belong to polar residues.     

Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.     

Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitor.

The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C(alpha)-C(beta) bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared.     

The crystal structure of guamerin in complex with chymotrypsin and the development of an elastase-specific inhibitor

Guamerin, a canonical serine protease inhibitor from Hirudo nipponia, was identified as an elastase-specific inhibitor and has potential application in various diseases caused by elevated elastase concentration. However, the application of guamerin is limited because it also shows inhibitory activity against other proteases. To improve the selectivity of guamerin as an elastase inhibitor, it is essential to understand the binding mode of the inhibitor to elastase and to other proteases. For this purpose, we determined the crystal structure of guamerin in complex with chymotrypsin at 2.5 Å resolution. The binding mode of guamerin on elastase was explored from the model structure of guamerin/elastase. Guamerin binds to the hydrophobic pocket of the protease in a substrate-like manner using its binding loop. In order to improve the binding selectivity of guamerin to elastase, several residues in the binding loop were mutated and the inhibitory activities of the mutants against elastase and chymotrypsin were monitored. The substitution of the Met36 residue for Ala in the P1 site increased the inhibitory activity against elastase up to 14-fold, while the same mutant showed 7-fold decreased activity against chymotrypsin compared to the wild-type guamerin. Furthermore, the M36A guamerin mutant more effectively protected endothelial cells against cell damage caused by elastase than the wild-type guamerin.     

Immunocytochemical evidence for a substance related to the bovine pancreatic polypeptide-peptide YY group of peptides in the human fetal gastrointestinal tract

The time of the first appearance and distribution of substance(s) reacting with the bovine pancreatic polypeptide (BPP) antiserum No. 146-6, i.e., BPP-like immunoreactivity, were studied in the gastrointestinal tract of 5-24-week-old human fetuses using an indirect immunoperoxidase method. The first immunostaining was identified at the 12th week of gestation in the oxyntic and colonic mucosa, and at the 10th week in the ileum. Serial sections alternately labelled with BPP and glicentin (GLI-1) antisera show several patterns. In the enteric and oxyntic mucosa, there is a cell population reacting only with the GLI-1 antiserum intermixed with cells containing both BPP-like and GLI-1-like immunoreactivities. In the oxyntic mucosa, however, certain cells might store BPP-like material only. Specificity tests illustrate cross-reactivity occurring in immunocytochemical studies of extrapancreatic BPP. The ability of synthetic BPP or a chemically related peptide, peptide YY to abolish the BPP antiserum immunoreaction, as well as previous radioimmunoassay data, raise the question of the presence of authentic BPP in GLI-1-containing cells.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation

The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.     

A new pyrimidine-specific ribonuclease from bovine seminal plasma that is active on both single and double-stranded polyribonucleotides and that can distinguish between Mg2+-containing and Mg2+-depleted naturally occurring RNAs

The purification to homogeneity of a new ribonuclease, named RNAase SPL, from bovine seminal plasma is described. This nuclease, like the bovine pancreatic RNAase A, is pyrimidine specific. Its activity on single-stranded synthetic polyribonucleotides such as poly(rU) is significantly higher than that of RNAase A. However, unlike RNAase A, RNAase SPL is highly active on a double-stranded RNA such as poly[r(A · U)], and shows extremely limited activity on naturally occurring RNAs, such as Escherichia coli RNA, prepared with Mg²⁺ present throughout the isolation procedure. Under conditions of limiting hydrolysis in which RNAase A degrades 60 to 90% of total E. coli RNA to acid-soluble material and the remaining to material having a molecular weight lower than that of transfer RNA, RNAase SPL does not yield any acid-soluble products: it does not appear to degrade tRNA or 5 S RNA, and causes only a small number of nicks in the remaining RNAs to yield a limiting digest containing products with molecular weights ranging between 10,000 and 150,000. Absence of Mg²⁺ during the isolation procedure, or heat denaturation of the RNA makes it as susceptible to RNAase SPL as it is to RNAase A.The above and other related observations reported here support the view that there are Mg²⁺-dependent structural features, besides single and doublestrandedness, in naturally occurring RNAs, that can be distinguished by using the two nucleases RNAase SPL and RNAase A.     

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.     

Enzymatic semisynthesis of aprotinin homologues mutated in P′ positions

The replacement of amino acids in the P₁ and P₂ position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the modified inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys¹⁵-Ala¹⁶, the residues P₁ (Ala¹⁶) and P₂ (Arg¹⁷) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a one pot reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala¹⁶-Arg¹⁷ was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg¹⁷-Ile¹⁸ peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys¹⁵ and Xaa¹⁶ are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P₁ residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.     

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.     

Structure of tick anticoagulant peptide at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at 1.6 A resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 A, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 A. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (Cys5T-Cys15T) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 A to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 A with the guanidinium group forming a cation-pi-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 A in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N-terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.