Substrate Specificity of 2-Ketogluc nate Reductase from Gluconobacti liquefaciens

The significant betaine aldehyde dehydrogenase activity was found in the cells of Pseudomonas aeruginosa A-16. The enzyme was inducibly formed and accumulated in the presence of choline, acetylcholine or betaine in the medium. The enzyme was purified approximately 620-fold with an overall recovery of 2.6% and proved to be homogeneous by ultracentrifugation. The molecular weight of the enzyme was determined as approximately 145,000 by gel filtration method. The enzyme had an isoelectric point around pH 5.1. The enzyme was quite specific for its substrate, betaine aldehyde. Both NADP and NAD functioned as coenzyme. The estimated values of Km at pH 7.4 and 25°C were 3.8 × 10^?4 m for betaine aldehyde, 8.9 × 10^?5 m for NADP and 2.2 × 10^?4 m for NAD.     

Amino Acid Residues Contributing to the Substrate Specificity of the Influenza A Virus Neuraminidase

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.     

Effects of 5,5'-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of ¹⁴C₂H₄ into the tissue and (b) the conversion of ¹⁴C₂H₄ to ¹⁴CO₂, was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to ¹⁴C₂H₄ markedly reduced both a and b. Increasing the ¹⁴C₂H₄ concentration from 0.14 to over 100 μl/l progressively increased the rate of a and b with tissue incorporation being greater than ¹⁴C₂H₄ to ¹⁴CO₂ conversion only below 0.3 μl/l ¹⁴C₂H₄. Reduction of the O₂ concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO₂ (5%) severely inhibited ¹⁴C₂H₄ to ¹⁴CO₂ conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during ¹⁴C₂H₄ treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.     

Substrate Specificity, Membrane Topology, and Activity Regulation of Human Alkaline Ceramidase 2 (ACER2)

Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Δypc1Δydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca²⁺ for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2ΔN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2ΔN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2ΔN13, but not ACER2ΔN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2ΔN13 or ACER2ΔN36 inhibited the glycosylation of integrin β1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca²⁺ for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.     

Molecular Analysis of Bacterial Community Based on 16S rDNA and Functional Genes in Activated Sludge Enriched with 2,4-Dichlorophenoxyacetic Acid (2,4-D) under Different Cultural Conditions

Differential emergence and diversity of bacterial communities from activated sludge in response to varied cultural conditions using 2,4-dichlorophenoxyacetic acid (2,4-D) were investigated by coupling molecular analyses based on 16S rDNA with functional genes. We employed three different cultural conditions: (1) a culture sequentially fed a high concentration (300 mg/L) of 2,4-D (HS); (2) a culture continuously fed a low concentration (10 mg/L) of 2,4-D (LC); and (3) a serial batch culture in which 1% (v/v) of culture was transferred to a fresh medium containing a high concentration (300 mg/L) of 2,4-D (HB). The HS and LC bioreactors were operated for 3 months and HB was repeatedly transferred for 1 month. The 2,4-D was stably degraded under all the cultural conditions tested. PCR amplification and cloning-based analysis of functional genes using community DNAs from the cultures revealed five different oxygenase genes that may be involved in the initial step of 2,4-D degradation. All five gene-types were present in HS, while one of the five genes, type V (tftA) was not detected in LC. Quantitative PCR analysis showed that in HS, Ralstonia eutropha JMP 134 type-tfdA4 (type I) was the most abundant in copy number (2.0 ± 0.1 × 10⁷ copies/g DNA) followed by RASC type-tfdA (type II) (1.8 ± 1.0 × 10⁶ copies/g DNA), putative cadA-like gene (type IV) (2.6 ± 0.8 × 10⁵ copies/g DNA), cadA gene (type III) (1.3 ± 1.0 × 10⁴ copies/g DNA), and tftA gene (type V) (3.5 ± 1.1 × 10³ copies/g DNA). Similar results were obtained in LC. In contrast, HB contained only type I and type III genes, and the type I gene was five orders of magnitude greater in copy number than the type III gene. Denaturing gel gradient electrophoresis (DGGE) analysis of PCR, amplified 16S rDNA fragments of bacterial communities in the three different cultures showed low similarity coefficient values (0.35) when compared to the original activated sludge, suggesting that 2,4-D amendment caused a drastic change in the bacterial community. Particularly, HB showed only six bands (16–18 bands in the other cultures) and very low similarity coefficient values when compared to the other communities (0.10 to HS, 0.17 to LC, and 0.0 to original sludge). These results indicated that serial batch culturing (HB) resulted in a phylogenetically limited number of 2,4-D degrading bacteria carrying limited catabolic genes whereas more diverse 2,4-D degraders and catabolic genes were present in HS and LC. Therefore, the approach used for monitoring should be taken into account when one evaluates the population dynamics of contaminant-degrading bacteria at bioremediation sites.     

Structural Insights into the Substrate Specificity of (S)-Ureidoglycolate Amidohydrolase and Its Comparison with Allantoate Amidohydrolase

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine into ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. In the final step of the pathway, (S)-ureidoglycolate amidohydrolase (UAH) catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, we report three crystal structures of Arabidopsis thaliana UAH (bound with substrate, reaction intermediate, and product) and a structure of Escherichia coli AAH complexed with allantoate. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-closed conformational transition in response to ligand binding and that the active-site size and the interaction environment in UAH and AAH are determinants of the substrate specificities of these two structurally homologous enzymes.     

Structural Basis of Fatty Acid Substrate Binding to Cyclooxygenase-2

The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H₂ from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co³⁺-protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 Å, respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted in AA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.     

Identification of Amino Acid Residues Required for the Substrate Specificity of Human and Mouse Chondroitin Sulfate Hydrolase (Conventional Hyaluronidase-4)

Human hyaluronidase-4 (hHYAL4), a member of the hyaluronidase family, has no hyaluronidase activity, but is a chondroitin sulfate (CS)-specific endo-β-N-acetylgalactosaminidase. The expression of hHYAL4 is not ubiquitous but restricted to placenta, skeletal muscle, and testis, suggesting that hHYAL4 is not involved in the systemic catabolism of CS, but rather has specific functions in particular organs or tissues. To elucidate the function of hyaluronidase-4 in vivo, mouse hyaluronidase-4 (mHyal4) was characterized. mHyal4 was also demonstrated to be a CS-specific endo-β-N-acetylgalactosaminidase. However, mHyal4 and hHYAL4 differed in the sulfate groups they recognized. Although hHYAL4 strongly preferred GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-containing sequences typical in CS-D, where GlcUA represents d-glucuronic acid, mHyal4 depolymerized various CS isoforms to a similar extent, suggesting broad substrate specificity. To identify the amino acid residues responsible for this difference, a series of human/mouse HYAL4 chimeric proteins and HYAL4 point mutants were generated, and their preference for substrates was investigated. A combination of the amino acid residues at 261–265 and glutamine at 305 was demonstrated to be essential for the enzymatic activity as well as substrate specificity of mHyal4.     

Response of Two Wheat Cultivars to CO(2) Enrichment under Subambient Oxygen Conditions

Two cultivars of wheat (Triticum aestivum L. cvs Sonoita and Yecora Rojo) were grown to maturity in a growth chamber within four sub-chambers under two CO₂ levels (350 or 1000 microliters per liter) at either ambient (21%) or low O₂ (5%). Growth analysis was used to characterize changes in plant carbon budgets imposed by the gas regimes. Large increases in leaf areas were seen in the low O₂ treatments, due primarily to a stimulation of tillering. Roots developed normally at 5% O₂. Seed development was inhibited by the subambient O₂ treatment, but this effect was overcome by CO₂ enrichment at 1000 microliters per liter. Dry matter accumulation and seed number responded differently to the gas treatments. The greatest dry matter production occurred in the low O₂, high CO₂ treatment, while the greatest seed production occurred in the ambient O₂, high CO₂ treatment. Growth and assimilation were stimulated more by either CO₂ enrichment or low O₂ in cv Yecora Rojo than in Sonoita. These experiments are the first to explore the effect of whole plant low O₂ treatments on growth and reproduction. The finding that CO₂ enrichment overcomes low O₂-induced sterility may help elucidate the nature of this effect.     

Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.     

Use of 4-Nitrophenoxyacetic Acid for Detection and Quantification of 2,4-Dichlorophenoxyacetic Acid (2,4-D)/(alpha)-Ketoglutarate Dioxygenase Activity in 2,4-D-Degrading Microorganisms

Purified 2,4-dichlorophenoxyacetic acid (2,4-D)/(alpha)-ketoglutarate dioxygenase (TfdA) was shown to use 4-nitrophenoxyacetic acid (K(infm) = 0.89 (plusmn) 0.04 mM, k(infcat) [catalytic constant] = 540 (plusmn) 10 min(sup-1)), producing intensely yellow 4-nitrophenol. This reagent was used to develop a rapid, continuous, colorimetric assay for the detection of TfdA and analogous activities in 2,4-D-degrading bacterial cells and extracts.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

Effect of N-tris (Hydroxymethyl) Methylglycine and N-2-Hydroxyethyl-piperazine-N'-2-Ethanesulfonic Acid Buffers on Linolenic Acid as a Substrate for Flaxseed Lipoxidase

The delay in, or loss of, flaxseed lipoxidase activity in N-tris (hydroxymethyl) methylglycine and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffers with linolenic acid as a substrate appears due to an alteration of the lipid micelle. Flaxseed lipoxidase activity is dependent on the ionic strength of the assay solution. These effects are not observed with linoleic acid as substrate. The influence of these 2 buffers on linolenic acid micelles may have a direct bearing on recent reports of chloroplast structure and activity in these buffers.     

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP₅) to inositol hexakisphosphate (IP₆). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP₅ in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in k_cat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP₄) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.     

Availability of O2 as a Substrate in the Cytoplasm of Bacteria under Aerobic and Microaerobic Conditions

The growth rates of Pseudomonas putida KT2442 and mt-2 on benzoate, 4-hydroxybenzoate, or 4-methylbenzoate showed an exponential decrease with decreasing oxygen tensions (partial O₂ tension [pO₂] values). The oxygen tensions resulting in half-maximal growth rates were in the range of 7 to 8 mbar of O₂ (corresponding to 7 to 8 μM O₂) (1 bar = 10⁵ Pa) for aromatic compounds, compared to 1 to 2 mbar for nonaromatic compounds like glucose or succinate. The decrease in the growth rates coincided with excretion of catechol or protocatechuate, suggesting that the activity of the corresponding oxygenases became limiting. The experiments directly establish that under aerobic and microaerobic conditions (about 10 mbar of O₂), the diffusion of O₂ into the cytoplasm occurs at high rates sufficient for catabolic processes. This is in agreement with calculated O₂ diffusion rates. Below 10 mbar of O₂, oxygen became limiting for the oxygenases, probably due to their high K_m values, but the diffusion of O₂ into the cytoplasm presumably should be sufficiently rapid to maintain ambient oxygen concentrations at oxygen tensions as low as 1 mbar of O₂. The consequences of this finding for the availability of O₂ as a substrate or as a regulatory signal in the cytoplasm of bacterial cells are discussed.     

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.     

Synthesis of a Glandular Secretion of the Civet Cat, (2S,6S)-(6-Methyltetrahydropyran-2-yl)acetic Acid and Its Enantiomer,by Using the Yeast-Reduction Product and Recovered Substrate from Yeast Reduction

A glandular secretion of the civet cat, (2S,6S)-(6-methyltetrahydropyran-2-yl)acetic acid 1 and its enantiomer, were synthesized from the yeast-reduction product and recovered substrate from yeast reduction.