How colipase-fatty acid interactions mediate adsorption of pancreatic lipase to interfaces

Colipase is a cofactor protein which forms a 1:1 complex with pancreatic lipase. This facilitates lipase adsorption to phosphatidylcholine-rich interfaces, presumably as a consequence of the higher affinity of colipase for such interfaces. According to this model, the presence of colipase in an interface should be sufficient to enable lipase adsorption from the aqueous phase. To test this hypothesis, mixed monolayers of colipase, phosphatidylcholine, and fatty acid at the argon-buffer interface were exposed to lipase injected into the stirred aqueous subphase. Spread colipase remained associated with the lipid monolayer in a surface pressure- and lipid composition-dependent manner. For example, with diacylphosphatidylcholine alone, colipase remained in the lipid monolayer at surface pressures 相似文献   

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.     

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity

Chemical modification studies and site-directed mutagenesis experiments have provided evidence that human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) are serine esterases. Loss of HLAL and HGL activity was also observed in the presence of sulfhydryl-reactive substances, suggesting that cysteines are likewise essential for substrate hydrolysis. To study the functional role of the HLAL and HGL cysteine residues, we replaced these amino acids with alanine by site-directed mutagenesis. Substitutions at positions 227 and 236, alone or together, drastically reduced hydrolytic activity in a substrate-dependent manner while the other mutants were not affected to any great extent. HLAL(Cys227-->Ala), HLAL(Cys236-->Ala), and HLAL(Cys227-->Ala, Cys236-->Ala) were essentially inactive against cholesteryl oleate, but retained about 23-39%, 28-37%, and 13-17% of catalytic activity for both triolein and tributyrin, respectively. The data obtained with the corresponding HGL mutants confirmed the importance of residues 227 and 236 in maintaining enzymatic activity towards long- and short-chain triglycerides. In order to assess the contribution of the eight amino acids delimited by Cys227 and Cys236 to lipolysis, we generated HLAL replacement mutants containing the corresponding residues 228-235 of HGL or RLL. Both HLAL chimeras were catalytically active towards all three substrates analyzed, indicating that these amino acids do not determine HLAL substrate specificity. Deletion of the eight-amino acid alpha-helix as well as disruption of its hydrophobic surface, in contrast, abolished enzymatic activity. Our studies suggest that Cys227, Cys236, and the amphipathic helix formed by residues 228-235 are essential for HLAL- and HGL-mediated neutral lipid catabolism.     

Structure and activity of rat pancreatic lipase-related protein 2

The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 A structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a beta-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.     

The affinities of procolipase and colipase for interfaces are regulated by lipids

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.     

Lipoxygenase products in human saliva: patients with oral cancer compared to controls

Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.     

Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: identification of the catalytically active serine, aspartic acid, and histidine residues

Human lysosomal acid lipase/cholesteryl ester hydrolase (HLAL), human gastric lipase (HGL), and rat lingual lipase (RLL) constitute a family of mammalian lipases characterized by an acidic pH optimum. HGL and RLL are secreted by the chief cells of the stomach and by the serous von Ebner's glands of the tongue, respectively, and hydrolyze dietary longchain triglycerides in the gastrointestinal tract. HLAL, in contrast, catalyzes the intralysosomal degradation of both triglycerides and cholesteryl esters in virtually all cells except erythrocytes. All three enzymes are proposed to be serine esterases with a catalytic Ser-Asp-His triad similar to other lipases, despite their sensitivity towards sulfhydryl modifying reagents. To investigate the role of conserved serine, aspartic acid, and histidine residues in HLAL and HGL, we constructed 24 mutant lipases with single amino acid substitutions using the site-directed mutagenesis approach. Our combined data strongly support the conclusion that Ser153, Asp324, and His355 are components of the catalytic triad of HLAL and HGL. Structural integrity of the conserved His-Gly dipeptide of lipases also appears to be important for neutral lipid hydrolysis, as replacement of His65 by glutamine abolished HLAL and HGL enzymic activity. Substitution of HLAL residues Asp93, Asp130, and Asp328 with glycine, in contrast, had a more pronounced impact on cholesteryl oleate hydrolysis than on triglyceride hydrolysis. These results provide new insights into the structural basis of HLAL and HGL function.     

Functional mapping of the surface residues of human thrombin

Utilizing site-directed mutagenesis, 77 charged and polar residues that are highly exposed on the surface of human thrombin were systematically substituted with alanine. Functional assays using thrombin mutants identified residues that were required for the recognition and cleavage of the procoagulant substrate fibrinogen (Lys21, Trp50, Lys52, Asn53 + Thr55, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Asp193 + Lys196, Glu202, Glu229, Arg233, Asp234) and the anticoagulant substrate protein C (Lys21, Trp50, Lys65, His66, Arg68, Tyr71, Arg73, Lys77, Lys106 + Lys107, Glu229, Arg233), interactions with the cofactor thrombomodulin (Gln24, Arg70) and inhibition by the thrombin aptamer, an oligonucleotide-based thrombin inhibitor (Lys65, His66, Arg70, Tyr71, Arg73). Although there is considerable overlap between the functional epitopes, distinct and specific residues with unique functions were identified. When the functional residues were mapped on the surface of thrombin, they were located on a single hemisphere of thrombin that included both the active site cleft and the highly basic exosite 1. No functional residues were located on the opposite face of thrombin. Residues with procoagulant or anticoagulant functions were not spatially separated but interdigitated with residues of opposite or shared function. Thus thrombin utilizes the same general surface for substrate recognition regardless of substrate function although the critical contact residues may vary.     

Anatomy of lipase binding sites: the scissile fatty acid binding site

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.     

Human milk lipases and their possible role in fat digestion

Human milk contains two lipases. One is a lipoprotein lipase with properties similar to the lipoprotein lipases that participate in the metabolism of blood plasma lipoproteins in several tissues. This enzyme is present in high activity in the lactating mammary gland where it facilitates the uptake of triglyceride fatty acids from the blood lipoproteins for production of milk lipids in the gland. The high activity of this enzyme in milk probably represent leakage of enzyme from the gland. This lipase is not stable at pH below 5 or in intestinal contents and it is unlikely that it participates in intestinal fat digestion. Its activity varies widely between individual milk samples, and there is a high correlation between its activity and the development of hydrolytic rancidity in the milk on storage. The other lipase is present in the milk in an inactive form which is activated by bile salts. This lipase is present in milk from primates but not in milk from lower animals. Human milk contains enough of this lipase to hydrolyze the milk lipids almost completely in less than half an hour at the pH and the bile acid and salt concentrations found in the small intestine of the human infant. It is probable that it increases the efficiency of milk fat absorption. The enzyme has a rather wide substrate specificity and may also act on other lipid substrates than triglycerides. In contrast to pancreatic lipase it hydrolyses all three ester bonds in a triglyceride. This may affect the physical chemistry of the lipids in the intestinal contents as well as their absorption and further metabolism in the musoca.     

Effects of muscle relaxants on catecholamine release from the bovine adrenal medulla

Studies on the structure and substrate specificity of purified rat kidney nuclear (RKN) lysozyme are reported. The carboxyl and amino terminal residues of RKN-lysozyme were found to be leucine and alanine respectively. The amino acid composition indicated similarities and differences as compared with that of hen egg white (HEW) lysozyme. There were alterations in the nine amino acid residues, Lys, His, Arg, Asp, Glu, Pro, 1/2 Cys, Tyr and Trp. The other nine residues were present in identical proportions to those of HEW-lysozyme. The decrease in the arginine and aspartic acid residues was found to be compensated by the increase in the number of lysine, histidine and glutamic acid residues. The overall ratio of the acidic to basic amino acids has thus been conserved in the mammalian enzyme. In addition, RKN-lysozyme contained decreased numbers of Trp, Tyr and 1/2 Cys, and increased numbers of proline residues as found in HEW-lysozyme. RKN-lysozyme did not cross react with heterologous antibodies produced against HEW-lysozyme, and vice versa. RKN-lysozyme showed distinct specificity towards the lysis of M. luteus. Against this substrate, it was three times more efficient than HEW-lysozyme. It also cleaved E. coli B, but its efficiency was half as much as with M. luteus. However, it cleaved P. septica and B. subtilis at a rate similar to HEW-lysozyme under identical conditions.     

Corandomization of fats improves absorption in rats

Human milk fat is well absorbed despite its large concentration of long-chain saturated fatty acids (LCSFA), particularly palmitic acid. The superior absorption has been ascribed in part to the uncommonly high proportion of the palmitic acid in the sn-2 position of the triglycerides, 70% in human milk triglycerides compared with 10-15% in the fats and oils commonly used in infant formula blends. Colipase-dependent pancreatic lipase selectively hydrolyzes the fatty acids at the sn-1 and 3 positions, yielding free fatty acids and the 2-monoglyceride, which are absorbable. Free palmitic acid, but not monopalmitin, can be lost as calcium soaps in the feces. The present study demonstrated that mixtures of coconut oil and palm olein are better absorbed by rats if the proportion of LCSFA in the sn-2 position is increased by the process of chemical randomization, in which the fatty acids of the native oils are redistributed equally to all three positions in the rearranged triglycerides. The fecal excretion of total fatty acids, essentially LCSFA, from the mixtures in which the oils were randomized together (corandomized) was 30 to 60% less than from the mixtures of native oils.     

Errors involved in instantaneous intravascular input assumptions

The triglyceride composition of rice lipids was investigated, using methods of thin-layer chromatography, enzymic hydrolysis by pancreatic lipase and gas-liquid chromatography. The major portion of triglycerides were tri- and di-unsaturated glycerides. The content of trisaturated glycerides was not more than 0.86%. The data obtained allow the conclusion that unsaturated acids occupy 2-positions in the triglyceride molecule of rice oil and oil of other plants.     

Effects of mutations in plastocyanin on the kinetics of the protein rearrangement gating the electron-transfer reaction with zinc cytochrome c. Analysis of the rearrangement pathway

We study, by flash kinetic spectrophotometry on the microsecond time scale, the effects of ionic strength and viscosity on the kinetics of oxidative quenching of the triplet state of zinc cytochrome c (3Zncyt) by the wild-type form and the following nine mutants of cupriplastocyanin: Leu12Glu, Leu12Asn, Phe35Tyr, Gln88Glu, Tyr83Phe, Tyr83His, Asp42Asn, Glu43Asn, and the double mutant Glu59Lys/Glu60Gln. The unimolecular rate constants for the quenching reactions within the persistent diprotein complex, which predominates at low ionic strengths, and within the transient diprotein complex, which is involved at higher ionic strengths, are equal irrespective of the mutation. Evidently, the two complexes are the same. In both reactions, the rate-limiting step is rearrangement of the diprotein complex from a configuration optimal for docking to the one optimal for the subsequent electron-transfer step, which is fast. We investigate the effects of plastocyanin mutations on this rearrangement, which gates the overall electron-transfer reaction. Conversion of the carboxylate anions into amide groups in the lower acidic cluster (residues 42 and 43), replacement of Tyr83 with other aromatic residues, and mutations in the hydrophobic patch in plastocyanin do not significantly affect the rearrangement. Conversion of a pair of carboxylate anions into a cationic and a neutral residue in the upper acidic cluster (residues 59 and 60) impedes the rearrangement. Creation of an anion at position 88, between the upper acidic cluster and the hydrophobic patch, facilitates the rearrangement. The rate constant for the rearrangement smoothly decreases as the solution viscosity increases, irrespective of the mutation. Fittings of this dependence to the modified Kramers's equation and to an empirical equation show that zinc cytochrome c follows the same trajectory on the surfaces of all the plastocyanin mutants but that the obstacles along the way vary as mutations alter the electrostatic potential. Mutations that affect protein association (i.e., change the binding constant) do not necessarily affect the reaction between the associated proteins (i.e., the rate constant) and vice versa. All of the kinetic and thermodynamic effects and noneffects of mutations consistently indicate that in the protein rearrangement the basic patch of zinc cytochrome c moves from a position between the two acidic clusters to a position at or near the upper acidic cluster.     

Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.     

Digestion and absorption of dietary triglycerides

This review is an attempt to put in order some facts obtained during twenty-five years of studying the digestion and absorption of dietary triglycerides. After a short history of this problem to show the progress of research, the active mechanism of pancreatic lipase on triglycerides is explained: this enzyme specifically hydrolyses fatty acids esterified with the primary hydroxyl groups of glycerol, forming 2-monoglycerides and free fatty acids. This fact is very important for the further process involving formation and composition of intraluminal micelles, along with absorption and de novo synthesis of triglycerides inside enterocytes. Several different points are disscussed: - Short or medium chain-length fatty acids are hydrolyzed more easily than long-chain ones; after their absorption, the former go to the liver by the portal circulation and the latter go to lymph. - An evaluation of the extent of digestive hydrolysis has been given by means of doubly-labelled triglycerides (glycerol and fatty acids), and by comparing isotope ratio values of lymph and dietary triglycerides. - The physico-chemical state that intraluminal fats are in is discussed; 2-monoglycerides and fatty acids released from triglyceride hydrolysis form macromolecular agregates with biliary salts and phospholipids, cholesterol, and dietary phospholipids (or their degradation products). These are termed mixed micelles and are absorbed by diffusion. - Many recent morphological studies carried out with the electron microscope are indicated. - Two metabolic pathways for triglyceride resynthesis in the mucosal cells have been established, one from free fatty acids and alpha-glycerophosphate, the other from 1 or 2-monoglycerides. Phosphatidic acids are involved in this resynthesis, but the role of other phospholipids is almost certainly not so narrowly limited. - The apparent digestibility of fats according to their fatty acid composition, and mainly according to the location of the fatty acids in the initial triglyceride molecule, is discussed. - The nature and importance of endogenous fatty acids in the digestive tract and lymph is discussed. - The lipid and fatty acid composition of lymph chylomicrons is given. - The importance of lymph lipid particles (average diameter 0,05 less than or equal mum) for transport of exogenous fatty acids is indicated. The biochemical mechanism involved in the digestion and absorption of dietary lipids are now well known, but some processes are still uncertain, particularly the physico-chemical state of intraluminal lipids, the role of enterocyte membranes, the synthesis and secretion of digestive enzymes, and the role of intestinal flora.     

Identification of residues lining the translocation pore of human AE1, plasma membrane anion exchange protein

AE1 is the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane. We have used scanning cysteine mutagenesis and sulfhydryl-specific chemistry to identify pore-lining residues in the Ser643-Ser690 region of the protein. The Ser643-Ser690 region spans transmembrane segment 8 of AE1 and surrounds Glu681, which may reside at the transmembrane permeability barrier. Glu681 also directly interacts with some anions during anion transport. The introduced cysteine mutants were expressed by transient transfection of HEK293 cells. Anion exchange activity was assessed by measurement of changes of intracellular pH, which follow transmembrane bicarbonate movement mediated by AE1. To identify residues that might form part of an aqueous transmembrane pore, we measured anion exchange activity of each introduced cysteine mutant before and after incubation with the sulfhydryl reagents para-chloromercuribenzene sulfonate and 2-(aminoethyl)methanethiosulfonate hydrobromide. Our data identified transmembrane mutants A666C, S667C, L669C, L673C, L677C, and L680C and intracellular mutants I684C and I688C that could be inhibited by sulfhydryl reagents and may therefore form a part of a transmembrane pore. These residues map to one face of a helical wheel plot. The ability to inhibit two intracellular mutants suggests that transmembrane helix 8 extends at least two helical turns beyond the intracellular membrane surface. The identified hydrophobic pore-lining residues (leucine, isoleucine, and alanine) may limit interactions with substrate anions.     

A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.     

Spectral diagnosis of tumor tissues by the method of fiber optic infrared spectroscopy]

Replacement of Trp39 of Rhizomucor pusillus pepsin (RMPP) by Asn or Cys resulted in a marked decrease in the milk-clotting and proteolytic activities. Kinetic analysis with chromogenic synthetic oligopeptides as substrates revealed that the mutations caused marked changes in the kcat value, but only slight changes in the Km value. Similar enzymatic properties were observed in mutants of Tyr75, which was shown to have a role in enhancing the catalytic activity. Both Tyr75Asn and Trp39Asn mutants rapidly lost the activity at high temperatures due to autocatalytic digestion at two sites. The structures of several aspartic proteinases including RMPP, as revealed by X-ray crystallographic studies, showed that Trp39 occupies a position close to Tyr75 and the N delta atom of Trp39 within hydrogen-bonding distance of the hydroxyl side chain of Tyr75. These observations suggest that Trp39 plays a role in maintaining Tyr75 in the correct orientation in aspartic proteinases, including RMPP.     

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.