Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂⁻ was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.     

Identification and biology of α-secretase

Our knowledge of the etiology of Alzheimer's disease (AD) has advanced tremendously since the discovery of amyloid beta (Aβ) aggregation in diseased brains. Accumulating evidence suggests that Aβ plays a causative role in AD. The β-secretase enzyme, beta-site APP cleaving enzyme-1 (BACE1), is also implicated in AD pathogenesis, given that BACE1 cleavage of amyloid precursor protein is the initiating step in the formation of Aβ. As a result, BACE1 inhibition has been branded as a potential AD therapy. In this study, we review the identification and basic characteristics of BACE1, as well as the progress in our understanding of BACE1 cell biology, substrates, and phenotypes of BACE1 knockout mice that are informative about the physiological functions of BACE1 beyond amyloid precursor protein cleavage. These data are crucial for predicting potential mechanism-based toxicity that would arise from inhibiting BACE1 for the treatment or prevention of AD.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline

Chaperone-like activities of α-crystallin, αB-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v(agg)) of UV-irradiated Phb on the concentration of α-crystallin or αB-crystallin is indicative of the existence of two types of chaperone-protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v(agg) on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggesting that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of α-crystallin and proline on aggregation of UV-irradiated Phb, a slight antagonism between proline used at a fixed concentration (0.15M) and α-crystallin was observed. At higher concentration of proline (0.5M) each chaperone acts independent of one another.     

Evaluation of Hydrophobicity Versus Chaperonelike Activity of Bovine αA- and αB-Crystallin

Calf lens αA-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than αB-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to αA-crystallin than to αB-crystallin at room temperature. Bis-ANS binding to both αA- and αB-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of αA-crystallin is lower than that of αB-crystallin whereas at higher temperatures, αA-crystallin shows significantly higher protection against aggregation of substrate proteins compared to αB-crystallin. Therefore, calf lens αA-crystallin is more hydrophobic than αB-crystallin and chaperone-like activity of α-crystallin subunits is not quantitatively related to their hydrophobicity.     

Metalloproteomics and metal toxicology of α-synuclein

Significant evidence has been accumulated linking exposure to heavy metals and/or distortion of metal homeostasis with the development of various neurodegenerative diseases. α-Synuclein is known to be involved in pathogenesis of a subset of neurodegenerative diseases collectively known as synucleinopathies. Therefore the interplay between metals, α-synuclein and neurodegeneration has attracted significant attention of researchers. This review discusses some of the aspects of the α-synuclein metalloproteomics and represents the peculiarities and consequences of α-synuclein interaction with various metal ions. Both non-specific and specific binding of this protein to metals is considered together with the analysis of the effects of such interactions on α-synuclein structure and aggregation propensity.     

Thermophilic biofiltration of methanol and α-pinene

Biofiltration systems utilizing thermophilic (55C) bacteria were constructed and tested for the removal of methanol and α-pinene — two important volatile organic compounds (VOCs) in the forest products industry. Thermophilic bacterial mixtures that can degrade both methanol and α-pinene were obtained via enrichment techniques. Two bench-scale thermophilic biofiltration systems (1085 and 1824 cm³) were used to examine compound removals at different residence times, with influent concentrations of 110 ppmv methanol and 15 ppmv α-pinene. At a residence time of 10.85 min, the smaller system had removal efficiencies of >98% for methanol, but only 23% for α-pinene. The larger system was operated with the same parameters to evaluate residence time and surfactant effects on compound removals. At a residence time of 18.24 min, both methanol and α-pinene removal rates were ≥95%. However, α-pinene removal dropped to 26% at a residence time of 6.08 min; methanol removal was not affected. Subsequent addition of a surfactant mixture increased α-pinene removal to 94% at the shortest residence time. No residual α-pinene was detected with the support medium Celite R-635, indicating that the surfactant may increase mass transfer of α-pinene. Journal of Industrial Microbiology & Biotechnology (2001) 26, 127–133. Received 06 June 2000/ Accepted in revised form 09 November 2000     

Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Molecular Divergence of Lysozymes and α-Lactalbumin

Abstract

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for α-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. α-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in α-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.     

Enzymatic Racemization of Leucine and α-Aminobutyrate

The distribution of amino acid racemase activities was investigated in the cell-free extracts of various strains of bacteria. Alanine racemase activity was exclusively found in all the strains tested. However, the cell-free extract of Strain 25-3, which has been identified as Pseudomonas striata, possessed the high activity catalyzing the racemization of alanine, α-aminobutyrate, leucine and methionine. The new and sensitive assay method of amino acid racemase with d-amino acid oxidase and 3-methyl-2-benzothiazolone hydrazone hydrochloride was established.

A new amino acid racemase catalyzing the conversion of either d or l enantiomorph of leucine and α-aminobutyrate to the racemates, was partially purified from the cell-free extract of Pseudomonas striata. Both the racemase reactions are suggested to be catalyzed by a single enzyme because of the constant ratio between the activities during the purification, and of their very resemble behavior to pH, temperature and heating the enzyme. Pyridoxal phosphate functions as the coenzyme for this racemase.     

Synthesis and Herbicidal Activities of α-Isocyanocycloalkylideneacetamides

Various α-isocyanocycloalkylideneacetamides were synthesized by the reaction of isocyanoacetamides with ketones and following by dehydration. These compounds were examined for their inhibitory activity against the germination of rice, cucumber and radish seeds, and for their herbicidal effects on rice, tomato and weed seedlings. Among them, α-isocyanocyclohexylidene-acetylpiperidine showed selective herbicidal activity against the broad-leaf plants.     

Structural and functional mapping of α-fetoprotein

Alpha-fetoprotein (AFP) is a major mammalian oncofetal protein, which is also present in small quantities in adults. It is a member of the albuminoid gene superfamily, which consists of AFP, serum albumin, vitamin D binding protein, and alpha-albumin (afamin). Although physicochemical and immunological properties of AFP have been well-studied, its biological role in embryo- and carcinogenesis and in adult organisms as well as mechanisms underlying its functioning remain unclear. During the recent decades, the biological role of AFP has been evaluated by identification of its functionally important sites. Comparison of primary structure of AFP and some physiologically active proteins revealed similarity of some polypeptide regions. This has been used for prediction of AFP functions (i.e., its multifunctionality). Localization of functionally important sites followed by determination of their amino acid composition and type of biological activity has provided valuable information for structural-functional mapping of AFP. Some peptide fragments of AFP have been synthesized and tested for biological activity. This review summarizes data on structural-functional interrelationships. We also describe functionally important AFP sites found by various groups during the last decade of structural-functional mapping of AFP with experimentally confirmed and putative biologically active sites.     

Expeditious and Stereoselective Synthesis of α-Guanosine

Abstract

The preparation of the unnatural nucleoside α-guanosine (1) has been achieved from readily available, literature precursors in ca. 6–9% overall yield. The key step, construction of the α-anomeric bond between the purine and the sugar, was accomplished by S_N2 displacement of protected β-chlororibose derivative 2 with 2-amino-6-chloropurine. The optimal conditions for this reaction involved cesium carbonate in N-methylpyrrolidinone (α/β ratio: 7.7:1).     

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H₂O₂ on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H₂O₂ and 0.1 mM FeCl₃ at 37 ^∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.     

Evaluation of Serum and Tissue Levels of α-Tocopherol

This study was designed to test the effect of supplementation of several antioxidants, including α-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for α-tocopherol by HPLC. Statistical increases in both serum and tissue α-tocopherol were found after supplementation. There was no statistical relationship between α-tocopherol and β-carotene levels.     

Isolation and Identification of α-Spinasterol and α-Spinasteryl D-Glucoside from Sugar Beet Pulp

A sterol and a steryl glucoside were isolated from dried beet pulp. The sterol was identified with α-spinasterol, the glucoside possessed chemical and physical properties such as follows: The molecular formula C₃₅H₅₈O₆, m.p. 292°, [ α]¹⁹_D-34.1°, acetate; m.p. 168°, benzoate; m.p. 175-177°, and positive for Molish and Lieber-mann-Burchard reactions. When it was hydrolyzed with 1% sulfuric acid, the crystal of α-spinasterol and D-glucose detectable by paper chromatography were obtained. These results gave evidence that the glucoside was in question to be α-spinasteryl D-glucoside.