Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties.

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.

Multiple ligand interaction of alpha-synuclein produced various forms of protein aggregates in the presence of Abeta25-35, copper, and eosin.

Various protein aggregates of alpha-synuclein developed by way of the common protein self-oligomerization in the presence of Abeta25-35, copper, and eosin were examined. All the aggregates exhibited congo red birefringence although the actual amounts of the aggregates were varied as determined by thioflavin T binding fluorescence. When their morphologies were analyzed in relation to in vitro cytotoxicity, the smallest granular aggregates obtained with copper exhibited the highest cytotoxicity, while the fibrous structures by eosin did not affect the cell.

Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein.

The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.

Self-oligomerization and protein aggregation of alpha-synuclein in the presence of Coomassie Brilliant Blue.

alpha-Synuclein has been implicated in various neurodegenerative disorders, including Parkinson's and Alzheimer's diseases, by its participation in abnormal protein depositions. As the protein has been suggested to play a significant role in the formation of the deposits which might be responsible for neurodegeneration, there is a strong demand to screen for alpha-synuclein-interactive small molecules. In this report, Coomassie Brilliant Blue (CBB) interaction of alpha-synuclein has been investigated with respect to induction of protein self-oligomerization in the presence of the chemical coupling reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Both CBB-G and CBB-R, which differ by only two methyl groups, induced the self-oligomerization of alpha-synuclein in a biphasic manner with optimal dye concentrations of 250 microM and 150 microM, respectively. The protein aggregates of alpha-synuclein induced by the dyes in the absence of the coupling reagent were analysed by electron microscopy. Whereas CBB-G induced formation of protein aggregates with a worm-like structure, CBB-R induced clear fibrilization of alpha-synuclein on a background of granular structures. CBB-R interacted with alpha-synuclein approximately twice as effectively as CBB-G (dissociation constants 0.63 microM and 1.37 microM, respectively). These dye interactions were independent from the acidic C-terminus of alpha-synuclein, which was reminiscent of the Alphabeta25-35 interaction of alpha-synuclein. However, the metal-catalysed oxidative self-oligomerization of alpha-synuclein in the presence of Cu2+/H2O2, which was augmented synergistically by Alphabeta25-35, was not affected by the dyes. This indicates that the dye binding site is also distinctive from the Alphabeta25-35 interaction site on alpha-synuclein. These biochemically specific interactions between alpha-synuclein and the dyes indicate that alpha-synuclein-interactive small molecules could provide a tool with which to approach development of diagnostic, preventive, or therapeutic strategies for various alpha-synuclein-related neurodegenerative disorders.

beta-catenin expression and mutational analysis in renal cell carcinomas.

beta-Catenin acts as a downstream transcriptional activator of the Wingless-Wnt signaling pathway. The beta-catenin-Tcf complex transactivates the downstream genes that regulate cell proliferation or inhibit apoptosis. The activation of this pathway through stabilization of beta-catenin is caused either by inactivating mutations of adenomatous polyposis coli (APC) tumor suppressor gene or by activating mutations in beta-catenin exon 3. To determine whether the abnormal expression and activating mutations in exon 3 of the beta-catenin gene are implicated in renal cell carcinogenesis, 52 renal cell carcinomas (RCC) were analyzed by immunohistochemistry, polymerase chain reaction-single-strand conformational polymorphism analysis (PCR-SSCP), and direct DNA sequencing. Immunohistochemically, all cases, as well as normal kidneys, showed membranous and/or cytoplasmic staining patterns without nuclear localization. However, the cytoplasmic accumulations of beta-catenin were observed in five (22.7%) of 22 cases of conventional (clear cell) renal carcinoma, but not in papillary or chromophobe renal carcinomas. The beta-catenin mutation was identified in only one case of conventional renal carcinoma and was a single-base missense mutation on codon 61, leading to substitution of glutamine by arginine. In conclusion, this study demonstrates that beta-catenin mutations are a relatively rare event in RCC and that cytoplasmic accumulations of beta-catenin protein are found only in conventional (clear cell) renal carcinomas. These data suggest that the activation of the beta-catenin signaling pathway may partly play a role in the development of conventional RCC.

Eosin interaction of alpha-synuclein leading to protein self-oligomerization.

Among various dyes including congo red, thioflavin S, thioflavin T, eosin, rhodamine 6G, and phenol red, the eosin was the only dye that induced self-oligomerization of alpha-synuclein in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline. To analyze chemical nature of the eosin interaction with alpha-synuclein, the phenomenon of self-oligomerization was further examined with eosin congeners such as ethyl eosin, eosin B, phloxine B, erythrosin B, and rose bengal. The followings are the conclusions we have reached. First of all, intactness of the benzoate moiety of eosin and the negative charge on the carboxylic group of the dye are important factors leading to the specific interaction with alpha-synuclein. Secondly, the localized negative charge on the xanthene moiety of eosin is another critical factor for the interaction. As far as substituting halides are concerned, bromides and iodides on the xanthene moiety of the dyes do not make any difference on the alpha-synuclein interaction because both eosin and erythrosin B have induced the common phenomenon of self-oligomerization. The binding curve between eosin and alpha-synuclein was sigmoidal as the dye concentrations were increased. A double reciprocal plot of the saturation curve showed that the maximum number of eosin binding sites on alpha-synuclein was 1.85 with a dissociation constant of 390 microM. The dye binding to the protein appeared to occur via a positive cooperativity. The eosin binding site(s) was suggested to be located predominantly on the NAC region and partly related to the acidic C-terminus of alpha-synuclein. It has been, therefore, expected that this information might be useful to develop alpha-synuclein interactive molecules, which could provide eventual preventive or possible therapeutic means against various alpha-synuclein related disorders including Parkinson's disease.

Metal-catalyzed oxidation of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide.

alpha-Synuclein is a component of abnormal protein depositions of Lewy bodies and senile plaques found in Parkinson's and Alzheimer's diseases, respectively. By using chemical coupling reagents such as dicyclohexylcarbodiimide or N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, the protein was shown to experience self-oligomerization in the presence of either copper(II) or Abeta25-35. The oligomers which appeared as a ladder on a 10-20% Tricine/SDS-PAGE have been suggested to participate in the formation of protein aggregations by possibly providing a nucleation center. Since oxidatively modified protein could increase its own tendency toward protein aggregation, metal-catalyzed oxidation of alpha-synuclein has been examined with copper(II) and hydrogen peroxide in the absence of the coupling reagent. Intriguingly, the protein was also self-oligomerized into an SDS-resistant ladder on the gel. This biochemically specific copper-mediated oxidative oligomerization was shown to be dependent upon the acidic C-terminus of alpha-synuclein because the C-terminally truncated proteins such as alpha-syn114 and alpha-syn97 were not affected by the metal and hydrogen peroxide. More importantly, the oxidative oligomerization was synergistically enhanced by the presence of Abeta25-35, indicating that the peptide interaction with alpha-synuclein facilitated the copper(II) binding to the acidic C-terminus and subsequent oxidative crosslinking. It has been, therefore, suggested that abnormalities in copper and H(2)O(2) homeostasis and certain pathological factors functionally similar to the Abeta25-35 could play critical roles in the metal-catalyzed oxidative oligomerization of alpha-synuclein, which may lead to possible protein aggregation and neurodegenerations.

Expression patterns of alpha-synuclein in human hematopoietic cells and in Drosophila at different developmental stages.

Alpha-synuclein, a presynaptic protein of the central nervous system, has been implicated in the synaptic events such as neuronal plasticity during development and learning, and neuronal degeneration under pathological conditions. As an effort to understand the biological function of alpha-synuclein, we examined the expression patterns of alpha-synuclein in various human hematopoietic cells, and in Drosophila at different developmental stages. The alpha-synuclein was ubiquitously expressed in all the tested hematopoietic cells including T cells, B cells, NK cells, and monocytes, as well as in the lymphoma cell lines, Jurkat and K562. A potential alpha-synuclein homologue was also expressed in Drosophila, and its expression appeared to be temporally and spatially regulated during development. Our data suggest that alpha-synuclein may function in invertebrates as well as in vertebrates and its function may not be restricted to the neuron.

Structural changes in alpha-synuclein affect its chaperone-like activity in vitro.

Alpha-synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of alpha-synuclein has not yet been established. We here present biochemical evidence that recombinant alpha-synuclein has a chaperone-like function against thermal and chemical stress in vitro. In our experiments, alpha-synuclein protected glutathione S-transferase (GST) and aldolase from heat-induced precipitation, and alpha-lactalbumin and bovine serum albumin from dithiothreitol (DTT)-induced precipitation like other molecular chaperones. Moreover, preheating of alpha-synuclein, which is believed to reorganize the molecular surface of alpha-synuclein, increased the chaperone-like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, alpha-synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone-like function of the protein. In addition, alpha-synuclein was also rapidly and significantly precipitated by heat in the presence of Zn2+ in vitro, whereas it was not affected by the presence of Ca2+ or Mg2+. Circular dichroism spectra confirmed that alpha-synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that alpha-synuclein could act as a molecular chaperone, and that the conformational change of the alpha-synuclein could explain the aggregation kinetics of alpha-synuclein, which may be related to the abolishment of the chaperonic-like activity.

Copper(II)-induced self-oligomerization of alpha-synuclein.

alpha-Synuclein is a component of the abnormal protein depositions in senile plaques and Lewy bodies of Alzheimer's disease (AD) and Parkinson's disease respectively. The protein was suggested to provide a possible nucleation centre for plaque formation in AD via selective interaction with amyloid beta/A4 protein (Abeta). We have shown previously that alpha-synuclein has experienced self-oligomerization when Abeta25-35 was present in an orientation-specific manner in the sequence. Here we examine this biochemically specific self-oligomerization with the use of various metals. Strikingly, copper(II) was the most effective metal ion affecting alpha-synuclein to form self-oligomers in the presence of coupling reagents such as dicyclohexylcarbodi-imide or N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The size distribution of the oligomers indicated that monomeric alpha-synuclein was oligomerized sequentially. The copper-induced oligomerization was shown to be suppressed as the acidic C-terminus of alpha-synuclein was truncated by treatment with endoproteinase Asp-N. In contrast, the Abeta25-35-induced oligomerizations of the intact and truncated forms of alpha-synuclein were not affected. This clearly indicated that the copper-induced oligomerization was dependent on the acidic C-terminal region and that its underlying biochemical mechanism was distinct from that of the Abeta25-35-induced oligomerization. Although the physiological or pathological relevance of the oligomerization remains currently elusive, the common outcome of alpha-synuclein on treatment with copper or Abeta25-35 might be useful in understanding neurodegenerative disorders in molecular terms. In addition, abnormal copper homoeostasis could be considered as one of the risk factors for the development of disorders such as AD or Parkinson's disease.

Comparisons of the NACP self-oligomerizations induced by Abeta25-35 in the presence of dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

NACP, the precursor protein of the non-amyloid beta/A4 protein (Abeta) component of Alzheimer's disease (AD) amyloid, also known as alpha-synuclein was shown to undergo self-oligomerization only in the presence of a modified Abeta fragment (residues 25 35) by using a relatively hydrophobic coupling reagent, dicyclohexylcarbodiimide (DCCD). Since the oligomerization not only required a relatively high concentration of DCCD but also its efficiency was suppressed even at a slightly basic pH of 7.5, another coupling reagent called N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was examined in order to make use of this technique to access the functional aspects of NACP in vitro by exploring more accurate and reproducible reaction conditions. The EEDQ also gave rise to the NACP oligomerization only in the presence of Abeta25-35 among the variously modified Abeta peptides. The reagent was about three times more effective than DCCD in terms of its optimal concentration to visualize the oligomers. In addition, its oligomerizing potency was not affected by the basic condition. Although physiological and pathological significance of the NACP self-oligomerization are currently unknown, this dramatic phenomenon and its visualization technique could shed light on the determination of molecular relationships of NACP with various intracellular or extracellular biomolecules related to the pathological conditions of Alzheimer's and Parkinson's diseases.

Epstein-Barr virus and CD21 expression in gastrointestinal tumors.

Epstein-Barr virus (EBV) was found in 7-17% of gastric adenocarcinomas, including lymphoepithelioma-like carcinomas, although its significance has not been clear. In addition, 20-30% of malignant lymphomas arising in the gastrointestinal tract have been known to express the EBV genome. Several lines of evidence indicate that EBV has been shown to infect both B lymphocytes and squamous epithelial cells via CD21 molecule in vivo and in vitro. The expression of CD21 in EBV-associated gastrointestinal tumors, however, has remained controversial. To determine the presence of CD21, an EBV receptor, in the EBV-associated gastrointestinal tumors, we, first, examined the EBV genome in sixty seven patients with either gastrointestinal adenocarcinomas or malignant lymphomas using in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and PCR for EBNA-1. Then, the investigation of CD21 expression was performed only in the EBV-positive tumors with immunohistochemical method for CD21 antigen on paraffin sections. EBERs were detected in 6 out of 26 gastric adenocarcinomas, 2 of 24 colonic adenocarcinomas, and 8 of 17 malignant lymphomas. EBERs were more prevalent in the malignant lymphomas originating from the small and large intestine (6/6) than from the stomach (2/11), and were detected in both B and T cell phenotypes. EBNA-1 was amplified in 11 of 16 EBERs-positive cases. Interestingly, however, none of the EBV-positive six gastric adenocarcinomas and eight malignant lymphomas expressed the CD21 on the cell surfaces or cytoplasm of both tumor cells and adjacent normal epithelial cells. These results suggest that EBV infection in the gastrointestinal malignancies would be mediated via different routes besides the CD21 or a new receptor distinct from CD21.

Self-oligomerization of NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease amyloid, observed in the presence of a C-terminal A beta fragment (residues 25-35).

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease (AD) amyloid, also known as alpha-synuclein, was suggested to seed amyloid plaque formation in AD by stimulating A beta aggregation. We have demonstrated that NACP experienced self-oligomerization only in the presence of a modified A beta fragment (A beta25-35) by using dicyclohexylcarbodiimide. This NACP oligomerization, appearing as a discrete ladder on a Tricine SDS-PAGE, was not observed with other A beta peptides such as the reverse peptide A beta35-25 and A beta1-40, indicating this process was specific not only for the C-terminal peptide sequence of the A beta but also for its orientation. It might be, therefore, suggested that the NACP self-oligomers formed only in the presence of a N-terminally truncated A beta peptide could act as a nucleation center for plaque formation during AD development.

Aluminum-induced structural alterations of the precursor of the non-A beta component of Alzheimer's disease amyloid.

The precursor of the non-A beta component of Alzheimer's disease amyloid (NACP) is a presynaptic protein whose function has been suspected to be tightly involved in neuronal biogenesis including synaptic regulations. NACP was suggested to seed the neuritic plaque formation in the presence of A beta during the development of Alzheimer's disease (AD). Recombinant NACP purified through heat treatment, DEAE-Sephacel anion-exchange, Sephacryl S-200 size-exclusion, and S-Sepharose cation-exchange chromatography steps appeared as a single band on SDS-PAGE with Mr of 19 kDa. Its N-terminal amino acid sequence clearly confirmed that the protein was NACP. Interestingly, however, the protein was split into a doublet on a nondenaturing (ND)-PAGE with equal intensities. The doublet was located slightly above a 45-kDa marker protein on a 12.5% ND-PAGE. In addition, the size of NACP was more carefully estimated as 53 kDa with high-performance gel-permeation chromatography using a TSK G3000sw size-exclusion column. Recently, Lansbury and his colleagues (Biochemistry 35, 13709-13715) have reported that NACP exists as an elongated "natively unfolded" structure which would make the protein more actively involved in protein-protein interactions and Kim (Mol. Cells 7, 78-83) has also shown that the natively unfolded protein is extremely sensitive to proteases. Here, we report that the structure of NACP could be altered by certain environmental factors. Aluminum, a suspected risk factor for AD, converged the doublet of NACP into a singlet with slightly lower mobility on ND-PAGE. Spectroscopic analysis employing uv absorption, intrinsic fluorescence, and circular dichroism indicated that NACP experienced the structural alterations in the presence of aluminum such as the secondary structure transition to generate about 33% alpha-helix. This altered structure of NACP became resistant to proteases such as trypsin, alpha-chymotrypsin, and calpain. Therefore, it is suggested that aluminum, which influences two pathologically critical processes in AD such as the protein turnover and the protein aggregation via the structural modifications, could participate in the disease.

Magnesium inhibits nickel-induced genotoxicity and formation of reactive oxygen.

Nickel compounds are recognized to cause nasal and lung cancers. Magnesium is an effective protector against nickel-induced carcinogenesis in vivo, although its mechanisms of protection remain elusive. The effects of magnesium carbonate on the cytotoxicity and genotoxicity induced by nickel subsulfide were examined with respect to the inhibition of cell proliferation, micronuclei formation, DNA-protein cross-link formation, and intranuclear nickel concentration. The generation of reactive oxygen by nickel chloride was also analyzed by observing 8-hydroxy-deoxyguanosine formation from deoxyguanosine in the presence and absence of magnesium chloride. The suppression of up to 64% of the proliferation of BALB/3T3 fibroblasts by nickel subsulfide (1 microgram/ml) was reversed by magnesium. The nickel compound increased not only the number of micronuclei but also the amount of DNA-protein cross-links examined with CHO and BALB/3T3 cells, respectively. These genotoxic effects of nickel were again lessened by magnesium carbonate. In addition, the cellular accumulation of nickel increased 80-fold with nickel subsulfide treatment and decreased with magnesium carbonate treatment. Nickel also enhanced 8-hydroxy-deoxyguanosine formation in the presence of H2O2 and ascorbic acid, where magnesium played another suppressive role. In fact, inhibition by magnesium was still observed even in the absence of nickel treatment. These results suggest that the protective role of magnesium in nickel-induced cytotoxicity and genotoxicity can be attributed to its ability to reduce either the intracellular nickel concentration or reactive oxygen formation.

Lowered temperature or binding of pyrophosphate to sites for noncatalytic nucleotides modulates the ATPase activity of the beef heart mitochondrial F1-ATPase by decreasing the affinity of a catalytic site for inhibitory MgADP.

Lineweaver-Burk plots for ATP hydrolysis catalyzed by bovine heart mitochondrial F1-ATPase (MF1) at 30 degrees C are biphasic, whereas they are linear at 15 degrees C. The rate of inactivation of the enzyme at 23 degrees C by 5'-[(p-fluorosulfonyl)benzoyl]adenosine (FSBA), which derivatizes noncatalytic nucleotide binding sites, is about 4 times faster when loss of activity is monitored at 15 degrees C as opposed to 30 degrees C. This suggests that maximal loss of ATPase monitored at 15 degrees C is observed when a single noncatalytic site is derivatized, whereas maximal inactivation at 30 degrees C requires modification of three noncatalytic sites. Prior incubation of MF1 depleted of endogenous nucleotides (nd-MF1) with pyrophosphate (PPi) stimulates ATPase activity 2-fold when assayed at 30 degrees C and pH 8.0. This stimulation correlates with binding of [32P]PPi to the second and third binding sites for PPi to be filled. Prior binding of PPi to nd-MF1 increases the rate of inactivation of the enzyme by FSBA at 23 degrees C about 4-fold when loss of activity is monitored at 30 degrees C and pH 8.0, whereas it does not affect the rate of inactivation when loss of ATPase is monitored at 15 degrees C or loss of ITPase is monitored at 30 degrees C. This indicates that the accelerated rate of inactivation induced by PPi when assays are conducted at 30 degrees C is not due to an increased rate of derivatization of noncatalytic sites. After 85% inactivation with FSBA, nd-MF1 retains the capacity to bind 2.8 mol of [32P]PPi per mole.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition and inactivation of the F1 adenosinetriphosphatase from Bacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide.

The F1-ATPase from Bacillus PS3 (TF1) hydrolyzes 50 microM ATP in three kinetic phases. An initial burst rapidly decelerates to a partially inhibited, intermediate phase, which, in turn, gradually accelerates to an uninhibited, final steady-state rate. Lauryl dimethylamine oxide (LDAO) stimulates the final rate over 4-fold. The stimulatory effect saturates at about 0.1% LDAO. Under these conditions, the intermediate phase is nearly absent. Dequalinium inhibits TF1 reversibly in the dark in the presence or absence of LDAO. The apparent affinity of TF1 for dequalinium increases in the presence of LDAO. Dixon plots of the initial rates of the intermediate phase and the final rates against dequalinium concentration at a series of fixed ATP concentrations in the presence and absence of 0.03% LDAO indicate noncompetitive inhibition in each case. Replots of the slopes of the Dixon plots for the initial rate of the intermediate phase and the final rate against 1/[ATP] reveal apparent Km values of 770 microM and 144 microM, respectively, when obtained in the absence of LDAO. The apparent Km values determined from the data obtained in the presence of LDAO for the same phases are 303 microM and 163 microM, respectively. These results suggest that LDAO stimulates ATPase activity either by increasing the affinity of noncatalytic sites for ATP, which promotes release of inhibitory MgADP from a catalytic site, or by directly promoting release of MgADP from the affected catalytic site. Dequalinium retards this process without affecting the affinity of noncatalytic sites for ATP. When irradiated in the presence of dequalinium, TF1 is rapidly inactivated with an apparent Kd of 12.5 microM in the presence or absence of LDAO.(ABSTRACT TRUNCATED AT 250 WORDS)

The TF1-ATPase and ATPase activities of assembled alpha 3 beta 3 gamma, alpha 3 beta 3 gamma delta, and alpha 3 beta 3 gamma epsilon complexes are stimulated by low and inhibited by high concentrations of rhodamine 6G whereas the dye only inhibits the alpha 3 beta 3, and alpha 3 beta 3 delta complexes.

The ATPase activity of the F1-ATPase from the thermophilic bacterium PS3 is stimulated at concentrations of rhodamine 6G up to about 10 microM where 70% stimulation is observed at 36 degrees C. Half maximal stimulation is observed at about 3 microM dye. At rhodamine 6G concentrations greater than 10 microM, ATPase activity declines with 50% inhibition observed at about 75 microM dye. The ATPase activities of the alpha 3 beta 3 gamma and alpha 3 beta 3 gamma delta complexes assembled from isolated subunits of TF1 expressed in E. coli deleted of the unc operon respond to increasing concentrations of rhodamine 6G nearly identically to the response of TF1. In contrast, the ATPase activities of the alpha 3 beta 3 and alpha 3 beta 3 delta complexes are only inhibited by rhodamine 6G with 50% inhibition observed, respectively, at 35 and 75 microM dye at 36 degrees C. The ATPase activity of TF1 is stimulated up to 4-fold by the neutral detergent, LDAO. In the presence of stimulating concentrations of LDAO, the ATPase activity of TF1 is no longer stimulated by rhodamine 6G, but rather, it is inhibited with 50% inhibition observed at about 30 microM dye at 30 degrees C. One interpretation of these results is that binding of rhodamine 6G to a high-affinity site on TF1 stimulates ATPase activity and unmasks a low-affinity, inhibitory site for the dye which is also exposed by LDAO.

Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.

Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional sites in F1-ATPases: location and interactions.

This review focuses on the location and interaction of three functional sites in F1-ATPases. These are catalytic sites which are located in beta subunits, noncatalytic nucleotide-binding sites which are located at interfaces of alpha and beta subunits and modulate the hydrolytic activity of the enzyme, and a site that binds inhibitory amphipathic cations which is at an interface of alpha and beta subunits. The latter site may participate in transmission of conformational signals between catalytic sites in F1 and the proton-conducting apparatus of F0 in the intact ATP synthases.