Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation

UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of beta-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.     

Functional properties of the glycosylated minor hemoglobins A1a-1,A1a-2 and A1b. EPR evidence for increased stability of the low affinity quaternary structure and decreased susceptibility to organic phosphate

Electron paramagnetic resonance (EPR) spectra of the glycosylated minor hemoglobins A1a-1, A1a-2, A1b and A1c and the major hemoglobin A0 in the nitrosyl form have been obtained in the absence and presence of inositol hexaphosphate. In the absence of inositol hexaphosphate, nitrosyl hemoglobins A1a-1, A1a-2 and A1b exhibited a triplet hyperfine structure centered at g = 2.009 which has been shown to be diagnostic of the low affinity (T) quaternary structure. Addition of inositol hexaphosphate to nitrosyl hemoglobins A0, A1c, A1b and A1a-2 developed a triplet hyperfine structure of the EPR spectra but the magnitude of the hyperfine was decreased in the order of hemoglobins A0, A1c, A1b and A1a-2. However, inositol hexaphosphate had essentially no effect on the EPR spectrum of nitrosyl hemoglobin A1a-1. The present results account qualitatively for the oxygen binding properties of these glycosylated minor hemoglobins in the framework of a two-state allosteric model.     

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production,Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.     

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.     

Cullin-4A·DNA damage-binding protein 1 E3 ligase complex targets tumor suppressor RASSF1A for degradation during mitosis

Tumor suppressor RASSF1A (RAS association domain family 1, isoform A) is known to play an important role in regulation of mitosis; however, little is known about how RASSF1A is regulated during the mitotic phase of the cell cycle. In the present study, we have identified Cullin-4A (CUL4A) as a novel E3 ligase for RASSF1A. Our results demonstrate that DNA damage-binding protein 1 (DDB1) functions as a substrate adaptor that directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex. Depletion of DDB1 also diminishes intracellular interactions between RASSF1A and CUL4A. Our results also show that RASSF1A interacts with DDB1 via a region containing amino acids 165-200, and deletion of this region abolishes RASSF1A and DDB1 interactions. We have found that CUL4A depletion results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of CUL4A and DDB1 markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We further show that CUL4A-mediated RASSF1A degradation occurs during mitosis, and depletion of CUL4A markedly reverses mitotic-phase-stimulated RASSF1A degradation. We also note that overexpression of CUL4A antagonizes the ability of RASSF1A to induce M-phase cell cycle arrest. Thus, our present study demonstrates that the CUL4A·DDB1 E3 complex is important for regulation of RASSF1A during mitosis, and it may contribute to inactivation of RASSF1A and promoting cell cycle progression.     

Frequencies of four genetic polymorphisms in the CYP1A2 gene in Turkish population

Cytochrome P450 (CYP) 1A2 gene is involved in the metabolic activation of several carcinogens and altered metabolization of some clinically used drugs. We aimed to investigate the distributions of genetic polymorphisms -3860 (G/A)(CYP1A2*1C) and -2467 (T/del)(CYP1A2*1D) in the 5'-flanking region and -739 (T/G)(CYP1A2*1E) and -163(C/A)(CYP1A2*1F) in the first intron of the CYP1A2 gene in 110 unrelated healthy Turkish volunteers by PCR-RFLP technique. The frequencies of each polymorphism in Turkish population were found as 0.04, 0.92, 0.01, 0.27 for CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, CYP1A2*1F, respectively. Compared with other populations, CYP1A2*1D has been found to be significantly increased in Turkish population. On the other hand, in general, the frequencies of the other polymorphisms were concordant with those in the Egyptian and Caucasian populations, and were different from those in the Japanese, Chinese and Ethiopian populations. Our results suggest that due to increased frequency of CYP1A2*1D in Turkish population, functional significance of CYP1A2*1D should be evaluated. It might be screened to determine the relationship between CYP1A2*1D and CYP1A2 related drug metabolisms in associated groups.     

Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1(336)) was previously shown to possess similar affinity for A2 and retain approximately 60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys(36) that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1(37-336)) showed little if any affinity for A2 (K(d)>2 microm), whereas factor VIIIa reconstituted with A2 plus A1(37-336)/A3-C1-C2 dimer demonstrated significant cofactor activity ( approximately 30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1(37-336) showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K(m) for factor X when A1 was cleaved at Arg(336). These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasma-based assays.     

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.     

Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Genetic Polymorphisms of Sulfotransferases (SULT1A1 and SULT1A2) in a Turkish Population

Sulfotransferases (SULTs) play a significant role in the biotransformation of a variety of xenobiotics and endogenous compounds. SULTs are genetically polymorphic enzymes; to date, 12 human cytosolic SULT isoforms have been identified. This study investigated SULT1A1 and SULT1A2 gene polymorphism using a PCR-RFLP method (n = 303). The frequency of the SULT1A1*1 allele was 76.2% and SULT1A1*2 was 23.8%. The SULT1A1*3 allele could not be identified. The SULT1A2 frequencies were 69.2% (SULT1A2*1), 18.3% (SULT1A2*2), and 12.5% (SULT1A2*3). The SULT1A1 and SULT1A2 loci were in Hardy-Weinberg equilibrium (SULT1A1 χ2 = 0.58, P = 0.44; SULT1A2 χ2 = 7.28, P = 0.06). Linkage analysis indicated a close linkage between these two genes (χ2 = 5.31, P < 0.01); therefore, the statistical hypothesis that SULT1A1 and SULT1A2 alleles are independently distributed was rejected. Additionally, a strongly positive linkage was detected between SULT1A1*2 and SULT1A2*2 alleles in this population (D' = 0.79, χ2 = 33.33).     

Induction of CYP1A by benzo[k]fluoranthene in human hepatocytes: CYP1A1 or CYP1A2?

While fresh human hepatocyte cultures are widely used to model hepatic cytochrome P450 (CYP) regulation and activity, their CYP1A subfamily composition induced by, e.g., polycyclic aromatic hydrocarbons is ambiguous. CYP1A1, CYP1A2, or both have been reported to be expressed, and their varied roles in chemical carcinogenesis makes resolution of which CYPs are expressed essential. We have used an immunoblot system with Bis-Tris-HCl-buffered polyacrylamide gel, which clearly resolves human CYP1A1 and CYP1A2, and polyclonal goat anti-human CYP1A1/CYP1A2 and rabbit anti-human CYP1A2 antibodies to probe the expressed CYP1A1 and CYP1A2 composition of seven individual human hepatocyte cultures induced with 5 microM benzo[k]fluoranthene (BKF) for 24 h. In six of the cultures only CYP1A1 was detected, and in the seventh both CYPs were detected. In most vehicle-treated hepatocyte cultures, neither CYP1A1 nor CYP1A2 was detected. In three additional hepatocyte cultures treated individually with BKF and 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD), the resultant induced CYP1A1/1A2 profiles were essentially not influenced by the nature of the inducing agents. To develop an activity-based assay to differentiate between CYP1A1 and CYP1A2 expression in human hepatocytes, our previously published R warfarin assay (Drug Metab. Disp. (1995) 23, 1339-1345) was applied to TCDD (10 nM)-treated hepatocyte culture. The low concentration of TCDD did not produce inhibition of the warfarin metabolism-such inhibition could confound the results. Based on the ratios of 6- to 8-hydroxywarfarin formed in two cultures, the ratios of CYP1A1/CYP1A2 expressed in these cultures were determined and they agreed with the ratios determined by immunoblot analysis. Thus each individual human hepatocyte culture must be characterized for induced CYP1A1 and CYP1A2 expression in studies of CYP1A activity. The warfarin assay provides a means of characterizing the cultures.     

Characterization of the function of the ver-1A and ver-1B genes, involved in aflatoxin biosynthesis in Aspergillus parasiticus.

The ver-1A gene was cloned and its nucleotide sequence was determined as part of a previous study on aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus SU-1. A second copy of this gene, ver-1B, was tentatively identified in this fungal strain. In this study, ver-1B was cloned by screening an A. parasiticus cosmid library with a ver-1A probe. The nucleotide sequence of ver-1B was determined. The predicted amino acid sequence of ver-1B had 95% identity with ver-1A. A translational stop codon, found in the ver-1B gene coding region, indicated that it encodes a truncated polypeptide. To confirm the function of the ver-1 genes in AFB1 synthesis, a plasmid (pDV-VA) was designed to disrupt ver-1A and/or ver-1B by transformation of the AFB1 producer A. parasiticus NR-1. One disruptant, VAD-102, which accumulated the pathway intermediate versicolorin A was obtained. Southern hybridization analysis of VAD-102 revealed that ver-1A but not ver-1B was disrupted. A functional ver-1A gene was transformed back into strain VAD-102. Transformants which received ver-1A produced AFB1, confirming that ver-1A is the only functional ver-1 gene in A. parasiticus SU-1 and that its gene product is involved in the conversion of versicolorin A to sterigmatocystin in AFB1 biosynthesis. A duplicated chromosomal region (approximately 12 kb) was identified upstream from ver-1A and ver-1B by Southern hybridization analysis. This duplicated region contained the aflR gene, which is proposed to be one regulator of AFB1, synthesis. A similar gene duplication was also identified in several other strains of A. parasiticus.     

Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mutagenesis and kinetic studies.

Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics. This reaction is catalyzed by a family of enzymes named sulfotransferases. The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities. SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine. In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3. First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols. The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds. When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine. The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached. Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine. The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates.     

Elongation Factor 1A Family Regulates the Recycling of the M4 Muscarinic Acetylcholine Receptor

In this study, we tested the hypothesis that the elongation 1A (eEF1A) family regulates the cell surface density of the M4 subtype of the muscarinic acetylcholine receptors (mAChR) following agonist-induced internalization. Here, we show that mouse brains lacking eEF1A2 have no detectable changes in M4 expression or localization. We, however, did discover that eEF1A1, the other eEF1A isoform, is expressed in adult neurons contrary to previous reports. This novel finding suggested that the lack of change in M4 expression and distribution in brains lacking eEF1A2 might be due to compensatory effects of eEF1A1. Supporting this theory, we demonstrate that the overexpression of either eEF1A1 or eEF1A2 inhibits M4 recovery to the cell surface after agonist-induced internalization in PC12 cells. Furthermore, eEF1A1 or eEF1A2 had no effect on the recovery of the M1 subtype in PC12 cells. These results demonstrate the novel ability of the eEF1A family to specifically regulate the M4 mAChR.     

Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.