Identification of glycated and acetylated lysine residues in human α2-antiplasmin

BackgroundThe post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues.AimWe sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin.MethodsWe analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of β-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM β-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer.ResultsEleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with β-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with β-d-glucose or ASA. Incubation with concomitant ASA and β-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM β-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM β-d-glucose was associated the absence of acetylation at that site.ConclusionHuman α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.     

Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Inhibitors of advanced glycation end product-associated protein cross-linking

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

HDAC3-dependent reversible lysine acetylation of cardiac myosin heavy chain isoforms modulates their enzymatic and motor activity

Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, PCAF, associate with cardiac sarcomeres, and a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study, we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to the A band of sarcomeres and was capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the K(m) for the actin-activated ATPase activity of both α- and β-MHC isoforms. By an in vitro motility assay, we found that lysine acetylation increased the actin sliding velocity of α-myosin by 20% and β-myosin by 36%, compared to their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli, independent of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.     

Sequence of amino acids comprising the single intra-chain disulfide loop in the alpha-chain of human fibrinogen.

Amino acid sequence studies have revealed that the largest cyanogen bromide fragment from the α-chain of human fibrinogen contains two cysteine residues which are situated thirty residues apart and near the carboxy-terminal end of that fragment. In contrast to a recent report, no other cysteines exist in this region of the α-chain. The sequence has been compared to disulfide loops in the β- and γ-chains and some very marginal homology suggested.     

Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris

A fibrinolytic enzyme has been purified from the fruiting bodies of Korean Cordyceps militaris. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fibrin-zymography, and gel filtration chromatography. The 15 amino acid residues of the N-terminal sequence of the enzyme were APVEQCDAPVGLARL, which is dissimilar to those of fibrinolytic enzymes from other mushrooms. Optimal pH and temperature values of the enzyme were 7.0 and 40°C, respectively. The enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), TPCK, 1,10-phenanthroline, Cu(2+), and Ba(2+). It was also significantly inhibited by aprotinin, EDTA, and EGTA. The enzyme showed a higher specificity for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, exhibiting that it is a chymotrypsin-like serine metalloprotease. The enzyme preferentially hydrolyzed the fibrinogen Aα-, followed by the Bβ-chains and the γ-chain. The α, β, and γ-γ chains of fibrin were also degraded by the enzyme.     

Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Inhibition of camel lens zeta-crystallin by aspirin and aspirin-like analgesics.

Camel lens zeta-crystallin was reversibly inhibited to various degrees by aspirin (acetyl salicylic acid) and the aspirin-like analgesics: paracetamol (acetaminophen) and ibuprofen (2-(4-isobutyl phenyl)-propionic acid). Among these, aspirin was the most potent inhibitor, causing nearly complete inhibition in a dose-dependent, but time-independent manner. Analysis of inhibition kinetics revealed that aspirin was uncompetitive inhibitor (K(i) 0.64 mM) with respect to NADPH and non-competitive inhibitor (K(i) 1.6 mM) with respect to the substrate, 9,10-phenanthrenequinone (PQ). Multiple-inhibition analysis showed that aspirin and pyridoxal 5' phosphate (PAL-P), a lysine specific reagent, simultaneously bound to a critical lysine residue located towards the NADPH binding region. Consistent with this, NADPH was able to substantially protect zeta-crystallin against aspirin, whereas PQ did not provide any protection. The results suggested that an essential lysine residue was the locus of aspirin binding. The inhibition of zeta-crystallin by aspirin and aspirin-like analgesics was reversible thus eliminating acetylation as a mechanism for inhibition. Reversible binding of aspirin to this lysine may cause steric hindrance resulting in uncompetitive inhibition with respect to NADPH.     

Chromatography and electron microscopy of cross-linked fibrin polymers--a new model describing the cross-linking at the DD-trans contact of the fibrin molecules

Polymerization of fibrin molecules results in the formation of a double-stranded protofibril. Although convincing data have not been presented, it is classically believed that γ-chain cross-linking of fibrin molecules occurs between the longitudinal end-to-end contacts (DD-long contacts) of the molecules within each of the two strands of a protofibril (intrastrand cross-linking). In this investigation the question addressed was whether γ-chain cross-linking takes place across the two strands (interstrand cross-linking) between the transversal half-staggered contacts of the molecules. Demonstration of double-stranded protofibrils in the presence of urea would indicate an interstrand cross-linking, whereas in the case of intrastrand cross-linking, the chaotropic agent urea would dissociate the double-stranded structure to form single-stranded fibrils. Protofibrils were obtained by generating soluble cross-linked fibrin polymers (sXLFbP): After incubation of souble fibrin polymers with Factor XIIIa at 37°C, the polymerization and cross-linking reaction was stopped by the addition of 6M urea and EDTA. Gel filtration of the reaction mixture in the presence of 3M urea was effect in separating sXLFbP from monomeric molecules. The sXLFbP-containing fractions were adsorbed onto mica in the presence of different concentrations of urea and investigated by electron microscopy after rotary shadowing. In the presence of 3M urea the sXLFbP appeared as double-stranded protofibrils. In the presence of 4M urea some parts of the double-stranded structure were found to be unfolded whereas in the presence of 6M urea multiple-bended single-stranded fibrils were observed. SDS-polyacrylamide gel electrophoresis of the sXLFbP demonstrated no γ-chain cross-linking within the protofibrils. Ultracentrifugation of the sXLFbP showed that in the presence of 3M urea noncross-linked fibrin polymers dissociated to monomeric molecules. When sXLFbP was centrifuged into 6M urea on sucrose density gradients, no reduction of the polymer size could be observed. The data indicate that γ-chain cross-linking occurs between the transversal contacts of the fibrin molecules within a protofibril, thus generating interstrand cross-linking. A model of the cross-linking of polymerized fibrin molecules is developed and the term DD-trans contact is proposed for this specific alignment of the D-domains.     

Identification of respective lysine donor and glutamine acceptor sites involved in factor XIIIa-catalyzed fibrin α chain cross-linking

Factor XIIIa-catalyzed ε-(γ-glutamyl)-lysyl bonds between glutamine and lysine residues on fibrin α and γ chains stabilize the fibrin clot and protect it from mechanical and proteolytic damage. The cross-linking of γ chains is known to involve the reciprocal linkages between Gln(398) and Lys(406). In α chains, however, the respective lysine and glutamine partners remain largely unknown. Traditional biochemical approaches have only identified the possible lysine donor and glutamine acceptor sites but have failed to define the respective relationships between them. Here, a differential mass spectrometry method was implemented to characterize cross-linked α chain peptides originating from native fibrin. Tryptic digests of fibrin that underwent differential cross-linking conditions were analyzed by high resolution Fourier transform mass spectrometry. Differential intensities associated with monoisotopic masses of cross-linked peptides were selected for further characterization. A fit-for-purpose algorithm was developed to assign cross-linked peptide pairs of fibrin α chains to the monoisotopic masses relying on accurate mass measurement as the primary criterion for identification. Equipped with hypothesized sequences, tandem mass spectrometry was then used to confirm the identities of the cross-linked peptides. In addition to the reciprocal cross-links between Gln(398) and Lys(406) on the γ chains of fibrin (the positive control of the study), nine specific cross-links (Gln(223)-Lys(508), Gln(223)-Lys(539), Gln(237)-Lys(418), Gln(237)-Lys(508), Gln(237)-Lys(539), Gln(237)-Lys(556), Gln(366)-Lys(539), Gln(563)-Lys(539), and Gln(563)-Lys(601)) on the α chains of fibrin were newly identified. These findings provide novel structural details with respect to the α chain cross-linking compared with earlier efforts.     

Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.     

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.     

Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth

Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between lysine acetylation and CMA.     

Interaction of α-N-acetyl-β-endorphin and calmodulin

Acetylation at the α-amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide β-endorphin, α-N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for α-N-acetyl-β-endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like β-endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to β-endorphin, suggest that residues 14–24 exhibit α-helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the α-amino terminal of β-endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, β-endorphin and the α-N-acetylated peptide behave very similarly with respect to calmodulin association.     

Antibodies Specific for α-N-Acetyl-β-Endorphins: Radioimmunoassays and Detection of Acetylated β-Endorphins in Pituitary Extracts

Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys⁹]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.     

Histone acetylation and reactive oxygen species are involved in the preprophase arrest induced by sodium butyrate in maize roots

Histone acetylation plays a critical role in controlling chromatin structure, and reactive oxygen species (ROS) are involved in cell cycle progression. To study the relationship between histone acetylation and cell cycle progression in plants, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor that can cause a significant increase in histone acetylation in both mammal and plant genomes, was applied to treat maize seedlings. The results showed that NaB had significant inhibition effects on different root zones at the tissue level and caused cell cycle arrest at preprophase in the root meristem zones. This effect was accompanied by a dramatic increase in the total level of acetylated lysine 9 on histone H3 (H3K9ac) and acetylated lysine 5 on histone H4 (H4K5ac). The exposure of maize roots in NaB led to a continuous rise of intracellular ROS concentration, accompanied by a higher electrolyte leakage ratio and malondialdehyde (MDA) relative value. The NaB-treated group displayed negative results in both TdT-mediated dUTP nick end labelling (TUNEL) and γ-H2AX immunostaining assays. The expression of topoisomerase genes was reduced after treatment with NaB. These results suggested that NaB increased the levels of H3K9ac and H4K5ac and could cause preprophase arrest accompanied with ROS formation leading to the inhibition of DNA topoisomerase.