Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and -ketoisocaproate (-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or -KIC on the oxidation of L-[U-¹⁴C]glutamate and L-[U-¹⁴C]glutamine in the brain were determined in the non-anesthetized rat. ¹⁴CO₂ generated by the metabolic oxidation of [^l4C]glutamate and [¹⁴C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and -KIC exhibited differential effects on ¹⁴CO₂ generation from radioactive glutamate or glutamine. Infusion of 0.5 mM -KIC increased [^l4C]glutamate oxidation approximately 2-fold; higher concentrations of -KIC did not further stimulate [¹⁴C]glutamate oxidation. The enhanced oxidation of [¹⁴C]glutamate may be attributed to the function of -KIC as a nitrogen acceptor from [¹⁴C]glutamate yielding [¹⁴C]-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [¹⁴C-]glutamine oxidation was not stimulated as much as [¹⁴C-]glutamate oxidation and only increased at 10 mM -KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [¹⁴C]glutamate or [¹⁴C]glutamine. In maple syrup urine disease elevated -KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.     

Ascorbic acid spares α-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells

Ascorbic acid, or vitamin C, can recycle -tocopherol in lipid bilayers, but even sparing of -tocopherol has not been a consistent finding in intact cells. Therefore, we tested the ability of ascorbate loading to spare -tocopherol and to prevent lipid peroxidation of cultured H4IIE rat liver cells. Although -tocopherol was undetectable in H4IIE cells, its cell content was increased by overnight incubation with -tocopherol in culture. Cells incubated with ascorbate 2-phosphate accumulated ascorbate to concentrations as high as 0.6 mM after overnight loading, but also released ascorbate into the medium. Ascorbate loading of -tocopherol-treated cells spared -tocopherol in a concentration-dependent manner during overnight incubation. Lipid peroxidative damage, measured as a decrease in fluorescence of cell-bound cis-parinaric acid, was decreased in cells loaded with either -tocopherol or ascorbate 2-phosphate, and showed an additive effect. These results suggest that ascorbate loading of H4IIE cells spares cellular -tocopherol and either directly or through recycling of -tocopherol prevents lipid peroxidative damage due to oxidant stress in culture.     

Primary structure of hemoglobin α-chain ofColumba livia (gray wild pigeon)

Primary structure of hemoglobin of -chain ofColumba livia is presented. The separation of -chain was obtained from globin by ion-exchange chromatography (CMC-52) and reversed-phase HPLC (RP-2 column). Amino acid sequence of intact as well as tryptic digested chain was determined on gas-phase sequencer. Structure is aligned homologously with 21 other species. Among different exchanges, positions 24 (TyrLeu), 26 (AlaGly), 32 (MetLeu), 64 (AspGlu), 113 (LeuPhe), and 129 (LeuVal) are unique to pigeon hemoglobin. The various exchanges in -chain are discussed with reference to evolution and phylogeny. The results show that the order Columbiformes is evolutionarily closer to the order Anseriformes. Since the pigeon is homogeneous, having HbA (^A-chain) and lacks ^D-chain, its phylogenetic placement could be established among birds having single hemoglobin components.     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Processing α-glucosidase I is an inverting glycosidase

-Glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc₃Man₉GlcNAc₂ to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the -glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(1-2)Glc(1-3)GlcO(CH₂)₄COOCH₃ was directly monitored by ¹H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released -glucose concomitant with the formation of the Glc(1-3)GlcO(CH₂)₈COOCH₃ disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.     

Binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to β-lactoglobulin: Influence of protein modifications

The binding of benzo()pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated -lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified -lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10^–8 M for ligand/-lactoglobulin complexes. The studied, chemically modified -lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of -NH₂ lysyl residues of -lactoglobulin increases the apparent molar ratios of benzo()pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native -lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand--lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of -lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo()pyrene, ellipticine, and cis-parinaric acid, respectively.     

Selective down-regulation of the Cqα/G11α G-protein family in tumour necrosis factor-α induced cell death

Investigations into the regulation of heterotrimeric GTP-binding protein a-subunits in models of tumour necrosis factor- (TNF)-induced cell death, revealed the selective down-regulation of the G_q/G₁₁ family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G_q/G₁₁ family protein levels, but not G_s, G_i and G_o protein levels as determined by Western analyses. This effect of TNF was observed in a concentration - and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G_q/G₁₁ family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca²⁺],_i release. In contrast, G_s activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca²⁺-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

Molecular properties of voltage-gated K+ channels

Subfamilies of voltage-activated K⁺ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K⁺ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K⁺ selectivity have been identified. Susceptibility of certain K⁺ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K⁺ channels from mammalian brain. These are large (M_r 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()₄()₄, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for ₁, ₂ and ₃ subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of ₁ and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K⁺ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins.     

Testosterone and progesterone metabolism in the central nervous system: Cellular localization and mechanism of control of the enzymes involved

Summary This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia.1. The activities of 5-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5-androstane-3,17-diol; 3-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5-reductase activity. A completely different localization was observed for 3-hydroxysteroid dehydrogenase; the formation of 3-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5-reductase and 3-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5-reductase, which metabolizes progesterone into 5-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-reduce progesterone. On the contrary, 3-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5-pregnane-3-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.     

Increased plasma α (1 → 3)-l-fucosyltransferase activities in patients with hepatocellular carcinoma

Alpha(1 3)-l-fucosyltransferase (1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma 1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma 1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma 1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma 1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.     

A method for the estimation of χ1 torsion angles in proteins

Summary A method for estimating CH-CH coupling constants from the shape and fine structure of NH-CH fingerprint-region cross peaks of COSY spectra is presented. Spectral simulations have been used to analyse the effect of variations in ³J_NH-CH, ³J_CH-CH, linewidths and digital resolution on the appearance of NH-CH COSY cross peaks. On the basis of these simulations a set of rules for broadly categorising experimental NH-CH cross peaks according to CH-CH coupling constants has been devised. The method has been applied to the analysis of NH-CH cross peaks of hen lysozyme. The results are compared to previous measurements of CH-CH coupling constants using E.COSY techniques.     

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis G subunit, GP1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNA^Arg _AGA/AGG. Isolation of the recombinant GP1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPS withan apparent K_d in the nM range. GTPS binding wasstimulated two-fold in the presence of Zn²⁺ compared with that inthe presence of Mg²⁺, Mn²⁺ or Ca²⁺.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPS,guanosine- 5-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGP1, recombinant GP1     

Identification of L-amino acid/L-lysine α-amino oxidase in mouse brain

Lysine, an essential amino acid is catabolized in brain through only the pipecolic acid pathway. During the formation of pipecolic acid, -deamination of lysine, and the formation of the -keto acid as well as its cyclized product are pre-requisites. The enzyme mediated -deamination of L-lysine and the formation of the -keto acid and the cyclized product are not demonstrated so far. Both lysine and pipecolic acid are known to increase in brain under the conditions of fasting, studies were therefore undertaken to identify the enzyme responsible for the -deamination of L-lysine in the brain tissue of mice which were fasted. The detection of the -keto acid of L-lysine, -keto--amino caproic acid and its cyclized product, ¹-piperidine-2-carboxylate was facilitated by the use of L-[U-¹⁴-C]-lysine as the substrate. The quantitation of the radioactivity in reaction products was done after separation by ion exchange chromatographic methods. The formation of the -keto acid was enzyme mediated, the -keto acid formed was established by reaction with N-methyl benzothiazolinone hydrazone hydrochloride. The cyclized product was accounted in a fraction which matched the resolution of authentic pipecolic acid on the Dowex column, and the cyclized product was confirmed by spectrophotometry. The hitherto undemonstrated -amino deaminating enzyme of L-lysine in brain tissue, the -keto acid of L-lysine and its cyclized product in a mammalian system could thus be demonstrated in the present study. These findings confirm the involvement of L-lysine oxidase/L-amino acid oxidase in the formation of pipecolic acid from L-lysine.     

SDS Induced Structural Changes in α-Crystallin and It’s Effect on Refolding

-Crystallin, a major eye lens protein and a key member of the small heat shock protein family, acts like a chaperone by preventing aggregation of substrate proteins. One of the hallmarks of most small heat shock proteins is their existence as a large oligomer, the role of which in its function is not understood at present. We have studied the role of the oligomer in the stability of its structure against SDS induced destabilization by CD measurements. -Crystallin from bovine source as well as recombinant preparation was used for this purpose. As SDS concentration was gradually increased, the -sheet structure was diminished followed by concomitant increase in the -helical structure. The quaternary structural changes in presence of SDS were also monitored by light scattering, polarization and anisotropy measurements. It was found that the breakdown of the oligomeric structure was nearly complete above 1 mM SDS concentration. The results were compared with that of a monomeric -crystallin, which is also a major -sheet protein like -crystallin. When -crystallin was first converted into monomeric random coil structure in presence of 6 M urea and allowed to refold in SDS solution, amount of -helix was more than that incubated directly in the same concentration of SDS. The results show that -crystallin attains extra structural stability against external stress due to its oligomeric structure. The implication for the extra stability is discussed in reference to its function as molecular chaperone.     

Phylogeny and evolutionary rates of G protein α subunit genes

Summary Rooted phylogenetic trees for a total of 34 genes encoding the stimulatory (s), inhibitory (i), transducin (t), G_x (x), G_z (z), G₁₁ (11), G₁₂ (12), G₁₃ (13), G₁₆ (16), G_q (q), and other (o) G protein a subunits have been constructed. The analysis shows that the G₁₂ (12 and 13), G_q (11, 16, and q), and G_s (s genes) groups form one cluster, and the G_x (x and z genes), G_i (i genes), G_t (t1 and t2), and G_o (o genes) groups form another cluster. During mammalian evolution, the rates of synonymous substitutions for these genes were estimated to be between 1.77 × 10^–9/site/year and 5.63 × 10^–9/site/year, whereas those of nonsynonymous substitutions were between 0.008 × 10^–9/site/year and 0.067 × 10^–9/site/year. These evolutionary rates are similar to those for histone genes, suggesting equally important biological functions of the G protein a subunits. Offprint requests to: S. Yokoyama