Flotillin-1 in the substantia nigra of the Parkinson brain and a predominant localization in catecholaminergic nerves in the rat brain.

The substantia nigra cells of a normal and Parkinson's disease human brain were obtained by the micropunch procedure and total RNA was isolated. Differential display RT-PCR of the total RNA revealed differentially expressed cDNAs that were identified by sequencing. This resulted in the identification of a panel of known and unknown differentially expressed genes. Complex I (NADH ubiquinone oxidoreductase) and Complex IV (cytochrome oxidase) whose expressions are decreased in Parkinson's disease were reduced in the Parkinson brain. Of the various differentially expressed genes, flotillin-1, also known as reggie-2, was of great interest to us. It is a relatively new protein which is an integral membrane component of lipid rafts and has been implicated in signal transduction pathway events. In situ hybridization histochemical studies with human and rat brain sections revealed the presence of this mRNA in discrete neuronal (and possibly glial) cells of the substantia nigra, locus coeruleus, cortex, hippocampus, hypothalamus, thalamus, motor nuclei, nucleus basalis, raphe nucleus, and other brain regions. Immunohistochemical studies revealed that flotillin-1 is not present in all the regions where the message was found. In the rat brain, the most prominent observation was the revelation of all catecholamine cells (dopamine, norepinephrine, epinephrine) by the flotillin-1 antibody (1:100 dilution). At a more concentrated dilution (1:10) other neuronal cells (e.g., cortex, thalamus, hindbrain) were observed. At both dilutions dense dopaminergic fibers were observed in the rat caudate-putamen, nigrostriatal tract, and substantia nigra. It is significant that there is an increased gene expression of flotillin-1 in the Parkinson substantia nigra/ventral tegmental area. The role of flotillin in these cells is unclear although it is interesting that the reggie-2/flotillin-1 gene was upregulated during retinal axon regeneration in the goldfish visual pathway (Schulte et al., Development 124:577-87, 1997) which suggests that flotillin-1/reggie-2 might play a role in axonal growth from the remaining substantia nigra cells of the Parkinson brain.

Pyruvate dehydrogenase E1 alpha isoform in rat testis: cDNA cloning, characterization, and biochemical comparison of the recombinant testis and liver enzymes.

Previous data indicated a tissue-specific regulation of mitochondrial pyruvate dehydrogenase (PDH) complex, especially in the brain and testis. The lack of biochemical data on the rat testis PDH limits comparative analysis between testis and liver enzymes. Therefore, we have isolated a cDNA clone encoding rat testis PDH E1 alpha isoform, determined its nucleotide sequence, studied the tissue-specific expression, and characterized the recombinant protein produced in bacteria, compared to the liver counterpart. Our cDNA clone (2.2 kb) contained the identical open reading frame (from nt 974 to 2149) with that previously reported (Cullingford et al., 1993 Biochim Biophys Acta 1216:149-153) but contained a long 5' untranslated region, which has little identity to the other clone. Northern blot confirmed testis-specific expression of this isoform. Genomic DNA analyses by PCR amplification suggested this clone is a gene product distinct from its X-linked somatic counterpart. Our biochemical and kinetic analyses revealed that the purified recombinant rat testis PDH E1 (containing both E1 alpha and E1 beta subunits) was enzymatically active and phosphorylated in vitro by purified PDH-kinase p48 or p45, similar to the recombinant human liver enzyme. Our current data thus indicate that the differential regulation of testis PDH observed in the animal model may result from differential modulation of PDH-kinase or -phosphatase in this tissue rather than the presence of functionally different PDH E1 subunit.

Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

Transcriptional induction of the cytochrome P4501A1 gene by a thiazolium compound, YH439.

The molecular mechanism of induction of cytochromes P4501A1/2 (CYP1A1/2) by a synthetic compound YH439 was studied in rodents as well as in cultured hepatoma cells. CYP1A1-mediated ethoxyresorufin-O-deethylase activity and amounts of its immunoreactive protein were increased in a time- and concentration-dependent manner after a single dose of YH439 (150 mg/kg). Northern blot analyses revealed that YH439 rapidly increased (< or = 2 hr) the levels of CYP1A1/2 mRNAs, resulting in an increase in CYP1A protein level by > 6-fold at 8 hr after injection. After YH439 administration, the levels of CYP1A1 and CYP1A2 mRNAs peaked at 8 hr and 16 hr, respectively, before returning to control levels at 16 and 24 hr. The CYP1A protein level, on the other hand, reached a maximum at 24 hr after YH439 treatment and returned to near-control levels at 72 hr. Nuclear run-on analyses revealed that YH439 induces CYP1A1/2 gene transcription as early as 2 hr after YH439 treatment. Cytosolic electrophoretic mobility shift assays suggested that YH439 activates the CYP1A1/2 genes through the aryl hydrocarbon (Ah) receptor and the xenobiotic response elements. The dependency on the Ah receptor for the induction of CYP1A1/2 by YH439 was confirmed by the lack of CYP1A1/2 induction in the Ah receptor knock-out mice (Ahr-1-) as well as in murine hepatoma cells without a functional Ah receptor. Molecular structural analysis of YH439 and several other compounds indicated that the planarity and size of a molecule are important in its interaction with the Ah receptor and subsequent CYP1A1/2 induction. YH439 is a thiazolium compound with little aromaticity and with a two-dimensional structure different from that of the Ahs. Therefore, it represents a new class of Ah receptor ligand and CYP1A inducer.

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N mutant.

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J.A., Gerlt, J.A., Kozarich, J.W., Koo, C.W., Shah, V.J., Kenyon, G.L., Neidhart, D.J., Fujita, J., & Petsko, G.A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R)- and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., Kenyon, G.L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.R., Petsko, G.A., & Kenyon, G.L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S)-atrolactate, with the exception that the side chain of His 297 is tilted and displaced approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S)-mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p-(bromomethyl)mandalate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pKa of His 297 in wild-type MR.

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the K166R mutant.

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts that either abstract the alpha-proton from the enantiomers of mandelate to form an enolic intermediate or protonate the enolic intermediate to form the enantiomers of mandelate, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. In this paper we report the structural and mechanistic properties of the mutant in which Lys 166 has been replaced with arginine (K166R). The structure of K166R has been determined at 1.85 A resolution with the substrate (S)-mandelate bound in the active site. The structure of this complex reveals no geometric alterations in the active site, with the exception that the longer side chain of Arg 166 is necessarily displaced upward from the position occupied by Lys 166 by steric interactions with the bound substrate. In contrast to the H297N mutant of MR [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9275-9281], the K166R exhibits low levels of racemase activity [kcat is reduced 5 x 10(3)-fold in the (R)- to (S)-direction and 1 x 10(3)-fold in the (S)- to (R)-direction]. The substrate and solvent deuterium isotope effects support a reaction coordinate for the K166R-catalyzed reaction in which the transition state for interconversion of bound (S)-mandelate and the stabilized enolic intermediate is higher in energy that the transition state for interconversion of bound (R)-mandelate and the stabilized enolic intermediate. The solvent deuterium isotope effect when (S)-mandelate is substrate (2.2 +/- 0.3) supports the proposal that the formation of the enolic intermediate involves partial transfer of a solvent-derived proton from Glu 317 to the substrate as the alpha-proton is abstracted [Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2777-2787].(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.

In the high-resolution X-ray structure of mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrogen-bonded to the carboxylate group of the bound inhibitor. This geometry suggests that the carboxylic acid functional group of Glu 317 participates as a general acid catalyst in the concerted general acid-general base catalyzed formation of a stabilized enolic tautomer of mandelic acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray structural analysis in the presence of (S)-atrolactate. No conformational alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for kcat were reduced 4.5 x 10(3)-fold for (R)-mandelate and 2.9 x 10(4)-fold for (S)-mandelate; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant.

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)