Protein nitration.

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 microM peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent Km of PKA for cAMP increased from approximately 10(-8) to 10(-6) M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.

Modification of proteins and polynucleotides by peroxynitrite.

Varied intensities of nitrotyrosine immunoreactivity were detected by Western blots after the reaction of proteins or enzymes with peroxynitrite (PN), a strong oxidant derived from nitric oxide. Intense immunoreactivity of cAMP-dependent protein kinase, calmodulin and most histones may depend on greater access to tyrosine residues in the reaction, whereas the absence of immunoreactivity of caspase-3, ubiquitin and S-100 proteins may reflect lack of accessibility. In addition, the changes in UV/visible absorbency were observed after PN-treatment of polynucleotides, polypeptides or proteins. Brief PN-treatment of invertase increased its enzymatic activity. Furthermore, PN-treatment of rabbit IgG decreased its recognition by anti-IgG. The results suggest that PN may chemically modify polypeptides, proteins and polynucleotides and may subsequently alter their biological activity.

Denitration of peroxynitrite-treated proteins by "protein nitratases" from dog prostate.

Putative "protein nitratases," which catalyze denitration of peroxynitrite (PN)-treated, proteins, were detected in the crude extract of dog prostate. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained prostate crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA, invertase, or polylysine. Nitratases were activated by preincubation with m-calpain/Ca2+. Furthermore, after denitration, the activity of PN/DTT-treated invertase decreased to the similar activity level of DTT-treated invertase. At least two different types of nitratases may occur: type I, reductant-dependent, and type II, reductant-independent.

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells.

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N6,O2-dibutyryl adenosine 3':5'-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12-72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35-110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus.

Denitration of peroxynitrite-treated proteins by 'protein nitratases' from rat brain and heart.

Putative 'protein nitratases,' which catalyze denitration of peroxynitrite (PN)-treated proteins, were detected in the homogenate/crude extract of rat brains and hearts. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained homogenate/crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA or invertase. Enhanced activity of nitratases was noted by preincubating crude extract with Ca2+. In addition, at least two types of nitratases may occur: type I, reductant-dependent, and type II, reductant- independent. Furthermore, upon denitration, the activity of PN-treated invertase increased to the same activity level of the untreated invertase. The overall reaction catalyzed by nitratases for denitration of nitrotyrosine residues in protein could be as follows: Protein-Tyr-NO2 + H2O --> Protein-Tyr-H + H+ + NO3-. The nitration/denitration of protein-tyrosine may be crucial in regulating signal transduction.

Interaction of matrix metalloproteinases-2 and -9 with pregnancy zone protein and alpha2-macroglobulin.

The binding of matrix metalloproteinases-2 and -9 to pregnancy zone protein and alpha2-macroglobulin was studied. The binding was demonstrated by formation of dimeric as well as tetrameric complexes of pregnancy zone protein and by the formation of alpha2-macroglobulin complexes with fast and intermediate mobility in native gel electrophoresis. The complex formation was confirmed by the use of 125I-labeled matrix metalloproteinase-2. The cleavage sites in the "bait" regions following formation of high-molecular-weight complexes of matrix metalloproteinases with the alpha-macroglobulins were determined by protein sequence analysis. Pregnancy zone protein was cleaved at Thr693-Tyr694 and alpha2-macroglobulin at Gly679-Leu680 and Arg696-Leu697 by matrix metalloproteinase-2. Matrix metalloproteinase-9 cleaved alpha2-macroglobulin at the same site as matrix metalloproteinase-2, but cleavage of pregnancy zone protein was at Leu753-Ser754. The sequences of the bands, visualized in the SDS gel, of approximately 90 and 165 kDa or higher molecular weight complexes were the same. This indicates that the matrix metalloproteinases cleaved the inhibitors with or without binding to them. The present results suggest that matrix metalloproteinases-2 and -9 may interact with pregnancy zone protein and alpha2-macroglobulin in vivo.

The contact zones in human alpha2-macroglobulin--functional domains important for the regulation of the trapping mechanism.

A functional domain termed the contact zone, which is the region of a subunit interacting with another non-covalently bound subunit, is suggested to play a decisive role in the trapping mechanism of human alpha2-macroglobulin. Tetrameric alpha2-macroglobulin can be dissociated into stable dimers with intact thiol esters by sodium thiocyanate, whereby the contact zones are disrupted. The dissociation leads to significant conformational changes, as studied by ultraviolet-difference spectroscopy, CD, fluorescence and affinity partitioning. The conformation of the dimers is similar to that of MeNH2-treated alpha2-macroglobulin, in which the thiol esters are cleaved, a conformational state with a closed trap occurs, and receptor-recognition sites are exposed. The receptor-binding domain is at least partly exposed in the dimer, as judged by binding of specific mAbs. The bait region in the dimers can be cleaved by proteases, and activation of the thiol esters ensues without binding of the protease. When the dimers were treated with MeNH2, no conformational changes could be detected by ultraviolet-difference spectroscopy or CD. The conformational changes occurring on dissociation into dimers are suggested to be related to trap closure and receptor-recognition-site exposure without cleavage of the thiol esters. The model presented here suggests that two separate conformational changes occur in alpha2-macroglobulin upon activation. The first involves changes at the contact zones as a result of the thiol-ester cleavage, and the second causes exposure of the receptor-recognition sites and closure of the trap.

Relationship between differentiation mechanisms involving cAMP-dependent protein kinase and protein kinase C in uninduced and differentiating HL-60 cells.

The influence of protein kinases on the differentiation of a human promyelocytic leukemia-cell line HL-60 to granulocytes was studied by using H8 and staurosporine as inhibitors of PKA and PKC respectively. In order to determine the significance of these protein kinases of uninduced and differentiating cells for the final differentiation, the cells were treated with the inhibitors before (-24 hours-0 hours) and after induction (0 hour-96 hours) of differentiation. To elucidate potential "cross-talking" between PKA and PKC of uninduced and differentiating HL-60 cells, the effects of H8 and staurosporine on differentiation were further examined by exposing the cells, pretreated with inhibitors for approximately one cell cycle time before induction, to the same or a different inhibitor. The results demonstrated that the effects of the inhibition of protein kinases of these cells on differentiation are protein kinase and inducer dependent. There is also inducer-dependent "cross-talk" between the differentiation mechanisms involving PKA and PKC activities in uninduced HL-60 cells and in cells induced to differentiate by dbcAMP or RA. However, the data also demonstrate that the PKC activity of uninduced cells involved in the differentiating mechanisms is not related to the PKC activity of dbcAMP-mediated differentiation and PKA is not related to PKC, respectively, in RA-mediated differentiation. The effects of the pretreatment of HL-60 cells with dbcAMP or RA for 24 hours on their subsequent differentiation induced by dbcAMP or RA are different. The pretreatment of cells with dbcAMP strongly potentiates RA-mediated differentiation, while the pretreatment with RA suppresses twofold dbcAMP-mediated differentiation.

The conformational state of human alpha 2-macroglobulin influences its dissociation into half-molecules by sodium thiocyanate.

Sodium thiocyanate dissociates native human alpha 2-macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2 M. Incubation with 1.6 M sodium thiocyanate for 1 h at 22 degrees C dissociates about 90% of alpha 2-macroglobulin into half-molecules. The half-molecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2 M or zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the "closed-trap" than in the "open-trap" conformation of alpha 2-macroglobulin. The cleavage in the bait region by trypsin makes alpha 2-macroglobulin completely stable toward dissociation, and alpha 2-macroglobulin remains in a tetrameric state in 2.2 M sodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation.

Preparation and characterization of a C-terminal fragment of pregnancy zone protein corresponding to the receptor-binding peptide from human alpha 2-macroglobulin.

Digestion of the pregnancy zone protein with papain at pH 4.5 yields an 18 kDa C-terminal fragment. This fragment consists of the 145 C-terminal amino-acid residues cleaved at Asn-1288 Ile and is homologous to the C-terminal receptor binding fragment of human alpha 2-macroglobulin obtained by cleavage with papain. The fragment contains an intrachain disulfide bond between 1308Cys and 1423Cys corresponding to that between 1304Cys and 1419Cys in alpha 2-macroglobulin. An oligosaccharide chain, is present in the C-terminal fragment of pregnancy zone protein as in human alpha 2-macroglobulin. The PZP C-terminal fragment was demonstrated to bind to the LRP/alpha 2M-receptor. Both the pregnancy zone protein and alpha 2-macroglobulin fragments bind three mAb's (alpha 1:1, R35, and 7H11D6) generated against alpha 2-macroglobulin. The mAb 7H11D6 was generated against the alpha 2-macroglobulin-proteinase complex (Isaacs, I.J., Steiner, J.P., Roche, P.A., Pizzo, S.V. and Strickland, D.K. (1988) J. Biol. Chem. 263, 6709-6714) and the binding of this to the C-terminal fragments of both pregnancy zone protein and alpha 2-macroglobulin indicates that both proteins use the same receptor recognition site for binding to the LRP/alpha 2M-receptor.

Protein kinase inhibitors exert stage specific and inducer dependent effects on HL-60 cell differentiation.

The human promyelocytic leukemia cell line HL-60 was induced to differentiate to mature granulocytic cells by dbcAMP or RA. The influence of distinct protein kinases during different stages of this differentiation was studied by the use of H8, staurosporine and genistein as inhibitors of PKA, PKC and PTK respectively. In dbcAMP-mediated differentiation, the PKA activity of uninduced cells is crucial for the induction of differentiation, but therefore its significance drastically declines and a more important role is played by PKC and PTK. In RA-mediated differentiation, the native state of PKA and PKC activities are necessary and of similar importance for induction. However, the differentiation is enhanced when, following induction, the activities of PKA and PTK are normal and the activity of PKC, in contrast, is temporary suppressed. At the phenotypic stage the effect of inhibition of protein kinases on maturation is in the order PTK > PKC > PKA for the dbcAMP-mediated differentiation and PKC > PKA > PTK for the RA-mediated differentiation. The results indicate that protein kinase activities during differentiation are stage specific and this specificity depends on the inducer used.

Methanethiolation of the liberated cysteine residues of human alpha 2-macroglobulin treated with methylamine generates a derivative with similar functional characteristics as native alpha 2-macroglobulin.

The thiol-modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human alpha 2-macroglobulin with methylamine. This methanethiolation generates a derivative of alpha 2-macroglobulin, with an 'open trap' and slow mobility in non-denaturing PAGE, similar to native alpha 2-macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native alpha 2-macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native alpha 2-macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice. In contrast to native alpha 2-macroglobulin, which can bind 2 mol chymotrypsin/mol, alpha 2-macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when alpha 2-macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group. The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half-life of 9 h as compared to a half-life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification.

Use of hydrophobic affinity partitioning as a method for studying various conformational states of the human alpha-macroglobulins.

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.

Probing different conformational states of pregnancy-zone protein. Fluorescence studies utilizing the binding of 4,4'-bis(8-anilino-1-naphthalenesulphonate).

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.

Comparison of conformational changes of pregnancy zone protein and human alpha 2-macroglobulin, a study using hydrophobic affinity partitioning.

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.

Ca2+ and pH dependence of hydrophobicity of alpha-lactalbumin: affinity partitioning of proteins in aqueous two-phase systems containing poly(ethylene glycol) esters of fatty acids.

Hydrophobic affinity partitioning in an aqueous two-phase system, composed of dextran and poly(ethylene glycol), has been used to study the hydrophobic binding capacity of bovine alpha-lactalbumin. The hydrophobicity of the poly(ethylene glycol)-containing phase was adjusted by including varying amounts of fatty acids bound to the polymer via an ester linkage. The change in the logarithmic partition coefficient of the protein in such systems was used as a measure of the hydrophobic binding. This value was strongly influenced by the amount of Ca2+ present as well as the pH value. The results are discussed in terms of the exposure of hydrophobic binding sites on alpha-lactalbumin and their relation to the conformational change in this protein due to Ca(2+)-binding, chelation of Ca2+ and pH dependence.

Influence of calmodulin on the human red cell membrane skeleton.

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.