alpha-chymotrypsin and methylamine

α2 -Macroglobulin (α2 M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2 M (hα2 M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2 M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.

Topics: alpha-Macroglobulins; Animals; Chickens; Chymotrypsin; Crystallization; Crystallography, X-Ray; Endopeptidases; Glycoside Hydrolases; Humans; Hydrolases; Methylamines; Peptide Hydrolases; Protease Inhibitors; Thermolysin

The stability of proteins is reduced by urea, which is methylamine and nonprotecting osmolyte; eventually urea destabilizes the activity and function and alters the structure of proteins, whereas the stability of proteins is raised by the osmolytes, which are not interfering with the functional activity of proteins. The deleterious effect of urea on proteins has been counteracted by methylamines (osmolytes), such as trimethylamine N-oxide (TMAO), betaine, and sarcosine. To distinctly enunciate the comparison of the counteracting effects between these methylamines on urea-induced denaturation of alpha-chymotrypsin (CT), we measured the hydrodynamic diameter (d(H)) and the thermodynamic properties (T(m), DeltaH, DeltaG(U), and DeltaC(p)) with dynamic light scattering (DLS) and differential scanning calorimeter (DSC), respectively. The present investigation compares the compatibility and counteracting hypothesis by determining the effects of methylamines and urea, as individual components and in combination at a concentration ratio of 1:2 (methylamine:urea) as well as various urea concentrations (0.5-5 M) in the presence of 1 M methylamine. The experimental results revealed that the naturally occurring osmolytes TMAO, betaine, and sarcosine strongly counteracted the urea actions on alpha-chymotrypsin. The results also indicated that TMAO counteracting the urea effects on CT was much stronger than betaine or sarcosine.

Topics: Calorimetry, Differential Scanning; Chymotrypsin; Light; Methylamines; Protein Conformation; Protein Denaturation; Protein Structure, Tertiary; Scattering, Radiation; Temperature; Thermodynamics; Urea

A new significantly improved method for purification of pregnancy zone protein (PZP), alpha 2-macroglobulin (alpha 2M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both alpha 2M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP-methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.

Topics: alpha-Macroglobulins; Binding Sites; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Cryopreservation; Electrophoresis, Polyacrylamide Gel; Female; Humans; Low Density Lipoprotein Receptor-Related Protein-1; Methylamines; Papain; Peptide Fragments; Pregnancy; Pregnancy Proteins; Protein Conformation; Receptors, Immunologic

The serum proteins alpha 2-macroglobulin and pregnancy zone protein undergo major conformational changes when complexed with proteinases. It is shown that the changes in delta log Kmax determined by hydrophobic affinity partitioning is a measure of the extent of changes in the conformation of these alpha-macroglobulins. We introduce a new term for the changes of surface hydrophobicity in a protein as delta log Kacc. This defines the difference of delta log Kmax between a modified and an unmodified conformational state of a specific protein and can be useful as a parameter to describe the apparent conformational changes in the protein.

Topics: alpha-Macroglobulins; Centrifugation; Chemical Phenomena; Chemistry, Physical; Chymotrypsin; Humans; Macromolecular Substances; Methyl Methanesulfonate; Methylamines; Polyethylene Glycols; Protein Conformation

Topics: alpha-Macroglobulins; Chymotrypsin; Humans; Immunoassay; Kinetics; Ligands; Methylamines; Phosphofructokinase-1; Polyethylene Glycols; Protein Binding; Protein Conformation; Proteins; Saccharomyces cerevisiae; Time Factors

Topics: alpha-Macroglobulins; Binding Sites; Chymotrypsin; Esters; Humans; Image Processing, Computer-Assisted; Methylamines; Microscopy, Electron; Models, Molecular; Molecular Conformation; Protease Inhibitors; Structure-Activity Relationship; Sulfhydryl Compounds

The alpha 1 proteinase inhibitor III from rat blood plasma, homologous to the alpha 2-macroglobulin family of proteins, has been studied in solution using small-angle scattering of X-rays and of neutrons: the radius of gyration, Rg, was found to be 4.5 nm, and the largest distance within the molecule, Dmax = 14 nm. When the inhibitor reacts with chymotrypsin or methylamine, the resulting derivatives yield slightly higher Rg-values, 4.7 and 4.85 nm, respectively. The data of the native protein are consistent with a model, the projection of which resembles the letter V and which is formed by the two identical halves of an elliptic cylinder with semi-axes of 2.1 and 5.5 nm and a length of 11 nm. This elliptic cylinder model also explained the scattering from the monomeric complement proteins C3 and C4, as well as that from the monomers of the dimeric and tetrameric alpha 2-macroglobulin family of proteins (Osterberg, R., et al. (1991), Biochemistry 30, 7873-7878). Due to the conformational change occurring when the thiol ester bond is split, the cleft in the V-form seems to be closed; and as a result, the models of the chymotrypsin and methylamine derivatives are more compact than that of the native protein.

Topics: Acute-Phase Proteins; Animals; Chemical Phenomena; Chemistry, Physical; Chymotrypsin; Computer Simulation; Male; Methylamines; Models, Chemical; Neutrons; Protease Inhibitors; Rats; Rats, Wistar; Scattering, Radiation; Solutions; X-Rays

Conformational changes of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP), reflected in changes in surface hydrophobicity, have been studied. The results show that the conformation of alpha 2M is governed by the degree of 'trapping'. Thus, cleavage in the bait region and of the thiol ester by proteinase treatment causes a two-fold increase in surface hydrophobicity of alpha 2M. However, the increase is still higher (three-fold) when the thiol esters in alpha 2M alone are cleaved by methylamine. Cyanylation of the thiol groups exposed upon methylamine treatment yields a derivative with the same hydrophobicity as native alpha 2M. Treatment of this derivative with chymotrypsin restores the hydrophobicity to that of methylamine-treated alpha 2M. Since the C-terminal 18 kDa fragment of alpha 2M exhibits no hydrophobicity, the change in hydrophobicity seems not to reside in the receptor binding site. In contrast to alpha 2M, modification of both native and methylamine-treated PZP with chymotrypsin gives a reduction (about 40%) in hydrophobicity. The change in hydrophobicity is insignificant on treatment with methylamine alone. Furthermore, hydrophobic interactions appear not to contribute to tetramerization of PZP. The present study indicates major differences in the conformational states of alpha 2M and PZP as reflected in the hydrophobic surfaces exhibited.

Topics: alpha-Macroglobulins; Chymotrypsin; Endopeptidases; Humans; Methylamines; Pregnancy Proteins; Protein Conformation; Solubility

The binding of the fluorescence probe 4,4'-bis(8-anilino-1-naphthalenesulphonate) (bis-ANS) to the human proteinase inhibitor pregnancy-zone protein (PZP) and its complexes with methylamine and chymotrypsin were investigated. The existence of dimeric PZP-chymotrypsin complex was demonstrated and both the dimeric and the tetrameric PZP-chymotrypsin complexes could be studied separately. The fluorescence data indicate that bis-ANS binds to two different sites on PZP and its complexes. The values of the dissociation constant, Kd1, for the binding to the high-affinity site were determined to be 231 +/- 14, 220 +/- 28, 114 +/- 15 and 49 +/- 1 nM, for the binding to native PZP, PZP-methylamine and dimeric and tetrameric PZP-chymotrypsin, respectively. An 11-30-fold decrease was observed in the affinity for the second site, the corresponding values of the dissociation constant, Kd2, being 1.5-2.8 +/- 1.0 microM, which are not significantly different for PZP and its derivatives. The results suggest that the probe bis-ANS discriminates between the different conformational states of PZP and that while the conformation of the complex with methylamine does not differ much from that of the native protein, there is a significant change in conformation when chymotrypsin cleaves the bait region. This is substantiated by a 30%-45% decrease in the maximum enhancement of fluorescence intensity when PZP is treated with chymotrypsin. Although the dimeric and tetrameric forms of PZP-chymotrypsin complexes differ in Kd1 values, the difference in the maximum enhancement of the fluorescence of bis-ANS by the two forms is not significant. This indicates that dimer-dimer interaction in the tetrameric form does not involve hydrophobic sites. The necessity of bait-region cleavage for extensive conformational changes in PZP distinguishes it from alpha 2-macroglobulin, the other alpha-macroglobulin in human plasma.

Topics: Anilino Naphthalenesulfonates; Chymotrypsin; Fluorescent Dyes; Humans; Methylamines; Pregnancy Proteins; Protein Conformation; Spectrometry, Fluorescence

Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.

Topics: alpha-Macroglobulins; Animals; Antibodies, Monoclonal; Chymotrypsin; Dinitrobenzenes; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Macromolecular Substances; Metabolic Clearance Rate; Methylamines; Mice; Pregnancy Proteins; Protease Inhibitors; Protein Conformation

Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma alpha 2-macroglobulin (alpha 2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the alpha 2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex alpha 2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of alpha 2M-methylamine from alpha 2M-chymotrypsin or alpha 2M-plasmin, a putative localization of the proteinases inside the alpha 2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form alpha 2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the alpha 2M-methylamine molecule and that the proteinases might be located in a very central position inside the alpha 2M-proteinase molecules.

Topics: alpha-Macroglobulins; Chymotrypsin; Fibrinolysin; Humans; Image Processing, Computer-Assisted; Methylamines; Microscopy, Electron; Protein Conformation; Thrombin; X-Ray Diffraction

Human alpha 2-macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines. The surface properties of the native, chymotrypsin-treated and methylamine-treated forms of alpha 2-macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two-phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of alpha 2-macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine-treated pregnancy zone protein, but the chymotrypsin-treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of alpha 2-macroglobulin and could be a signal for binding to cell-surface receptors, followed by internalization.

Topics: alpha-Macroglobulins; Centrifugation; Chemical Phenomena; Chemistry, Physical; Chymotrypsin; Dextrans; Humans; Methylamines; Palmitic Acid; Palmitic Acids; Polyethylene Glycols; Pregnancy Proteins; Surface Properties

The cellular binding and uptake was studied for alpha 1-inhibitor3, a monomeric 200 kDa proteinase inhibitor present in rat plasma. After intravenous injection in the rat the inhibitor disappeared from the circulation with a half-time of 2.5 min when complexed with chymotrypsin, whereas the half-time for uncomplexed inhibitor was more than 60 min. 6 min after the injection of labelled complex, 83% was in the liver and 2.5% in the spleen. In vitro experiments at 4 degrees C with isolated hepatocytes and peritoneal macrophages showed binding to the previously described receptors which bind and internalize the tetrameric rat and human alpha 2-macroglobulin-proteinase complexes. The binding affinities were similar for the two types of complexes and binding was followed by uptake and degradation of the labelled complex when the cells were warmed to 37 degrees C. The binding of uncomplexed alpha 1-inhibitor3 was low and did not increase following treatment with methylamine in spite of cleavage of the internal thiol ester. alpha 1-Inhibitor3-methylamine was changed to the receptor binding form when treated with chymotrypsin which caused the cleavage of at least one peptide bond in the bait region.

Topics: Acute-Phase Proteins; alpha-Macroglobulins; Animals; Chymotrypsin; Half-Life; Humans; Liver; Low Density Lipoprotein Receptor-Related Protein-1; Male; Methylamines; Protease Inhibitors; Rats; Rats, Inbred Strains; Receptors, Immunologic; Spleen; Trypsin

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.

Topics: alpha-Macroglobulins; Chymotrypsin; Electron Spin Resonance Spectroscopy; Humans; Methylamines; Models, Theoretical; Molecular Weight; Protein Binding; Protein Conformation

The structural change that occurs in alpha-2-macroglobulin upon its interaction with methylamine or chymotrypsin was studied by high-performance gel chromatography and electron microscopy. The result enabled us to estimate the Stokes radius of the protein as 8.8 nm and 7.9 nm before and after binding with the proteinase, respectively. The methylamine-treated protein also had the Stokes radius of 7.9 nm. Similar studies on the chicken and crocodilian ovomacroglobulins showed that these homologues of alpha 2-macroglobulin had Stokes radii of 9.2-9.3 nm and 8.5-8.7 nm before and after binding with chymotrypsin. Their Stokes radii did not change as a result of the methylamine treatment. Electron micrographs of the native and altered forms of the three proteins are presented. This study introduces a simple and quantitative method to study the structural change of alpha 2-macroglobulin and its homologues.

Topics: Alligators and Crocodiles; alpha-Macroglobulins; Animals; Chickens; Chromatography, Gel; Chymotrypsin; Female; Humans; Macroglobulins; Male; Methylamines; Microscopy, Electron; Protein Conformation

Analogues of the microbial proteinase inhibitor chymostatin have been synthesized. The two most promising analogues were tested on protein turnover in isolated rat hepatocytes. Their effect is much similar to the effect of chymostatin, but the analogues are even more powerful inhibitors, probably due to an increased effect on lysosomal thiol proteinases. The analogues blocked most of the lysosomal (i.e. methylamine-sensitive) degradation of endogenous protein and caused a 50% inhibition of the non-lysosomal degradation; the effect occurred rapidly and was reversed upon washing the cells. One of the analogues, Z-Arg-Leu-Phe(H), is the most potent inhibitor of hepatic protein degradation so far found.

Topics: alpha-Fetoproteins; Animals; Asialoglycoproteins; Chymotrypsin; Fetuins; Liver; Male; Methylamines; Oligopeptides; Proteins; Rats; Rats, Inbred Strains; Time Factors