Intracellular pH during daily torpor inPeromyscus maniculatus

Summary Intracellular and extracellular acid-base parameters during normothermy and daily torpor were examined in deer mice (Peromyscus maniculatus). [¹⁴C]Dimethyloxazolidinedione and [³]inulin were used to assess intracellular pH in liver, heart, skeletal muscle, and brain. Buffering capacities were determined using tissue homogenates. A significant increase in plasma and during daily torpor indicates a respiratory acidosis. All tissues experienced a reduction in the calculated dissociation ratio of histidine imidazole groups (_imid) during daily torpor (16.5% for brain, approximately 10% for other tissues). Based on comparisons with physicochemical tissue buffering capacities, metabolic compensation of the respiratory acidosis occurred in liver, heart, and plasma, while brain was more acidotic than predicted. The more extensive change in brain _imid might influence a regulated decrease in body temperature. Comparison of acid-base parameters during daily torpor and hibernation suggests that the magnitude of acid-base modifications in mammals may be associated with the level of dormancy.Abbreviations _imid dissociation ratio of histidine imidazole groups - physicochemical non-bicarbonate buffer value - ' apparent (in vivo) buffer value - bicarbonate bicarbonate values corrected to a temperature of 25 °C - pH pH values corrected to a temperature of 25 °C - pH _i intracellular pH - pK _imid pK of histidine imidazole groups - T _b body temperature     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

Na+-transport modulation induces isoform-specific expression of Na+,K+-ATPase α-subunit isoforms in C2C12 skeletal muscle cell

Changes in demands for Na⁺ transport alter expression of the Na⁺,K⁺-ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the 2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the -subunit isoforms by Na⁺ in the C₂C₁₂ skeletal muscle cell that expresses the 1 and 2 isoforms. Western blot analysis showed that in differentiating C₂C₁₂ muscle cell, but not in undifferentiated myoblast, veratridine, a Na⁺ channel activator, greatly increased expression of the 2 isoform; expression of 1 was unaltered. Because the level of -actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na⁺ transport by tetrodotoxin again failed to alter expression of a1. Thus, in C₂C₁₂ skeletal muscle cell, changes in Na⁺ transport alters expression of the 2, but not the 1 isoform. These results differ from those observed previously in muscle cells that express only the 1 isoform. Because mammalian skeletal muscle expresses both the 1- and 2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Purification and characterization of biliverdin IXalpha from Atlantic salmon (Salmo salar) bile

Biliverdin IX was purified from the bile of Atlantic salmon (Salmo salar) using a silica gel (Wakogel C-200) column. The yield was 49.5 mg per 100 ml of fresh bile and purity 95.3%. The biliverdin IX in the bile was quite stable when the bile was frozen at –80°C for a period of 40 days. However, 7.1% of the biliverdin IX was lost when the bile was stored at 4°C for 20 days. The purified biliverdin IX appeared as a single spot with R_f value of 0.25-0.27 on thin layer chromatography (TLC) and one main peak on high performance liquid chromatography (HPLC) at 436 or 650 nm. When the biliverdin IX was subjected to enzymic reduction with highly purified biliverdin reductase, two clear isobestic points were seen, at 384 and 670 nm. When the products of the reaction with biliverdin IX were extracted in butanol after completion of the reaction, one absorbance peak was observed at 468 nm. The time course of the reduction of biliverdin IX to bilirubin IX catalyzed by biliverdin reductase depended on reduced pyridine nucleotide. The time course of the NADPH-dependent reaction is different from that of the reaction with NADH. In the reduction of biliverdin IX , per mole of biliverdin IX reduced or per mole of bilirubin IX formed 1 mole of reduced pyridine nucleotide was consumed in both the NADH and NADPH systems.     

Modification of lignin by Geotrichum klebahnii

¹³C-NMR spectroscopic analysis indicates that the yeast-like species Geotrichum klebahnii is an efficient microorganism for lignin biodegradation. This strain modified beechwood lignin even if it was the only carbon source by C-C side chain cleavage, C-oxidations, aromatic ring cleavage and reductive reactions. The obtained results outline prospective application of G. klebahnii for biotechnological pre-treatment of lignocellulosic materials.     

Identification of functionally important cysteines in the alpha-subunit of transducin by chemical cross-linking techniques

Transducin (T), the G-protein in the visual system, is a heterotrimer arranged as two functional units, T and T. N, N-1, 2-phenylenedimaleimide (o-PDM) and N, N-1, 4-phenylenedimaleimide (p-PDM), two cysteine specific-homobifunctional agents, were used to covalently cross-link T and its units. A complete inhibition in T function was observed in the presence of these compounds. Incubation of T with o-PDM or p-PDM resulted in the formation of high-molecular-weight oligomers of 70-, 105-, 140-, and 200 kDa, as well as intramolecular cross-linked polypeptides that migrated as 35- and 37-kDa bands. Additionally, the treatment of T with both reagents produced a major species of 46-kDa. The combination of intact T and o-PDM- or p-PDM-treated T reconstituted T native activities. On the contrary, when o-PDM- or p-PDM-modified T was incubated with intact T, more than 90% inhibition on T function was observed. Hence, the cysteines modified and/or cross-linked on T represent functionally important residues of T.     

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium

Summary Regulation of Na,K-ATPase mRNA isoform and mRNA expression by thyroid hormone (T₃) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA₁, mRNA₃, and mRNA₁ abundances were nearly constant while mRNA₂ was undetectable. During the interval between postnatal days 5 and 15, mRNA₃ decreased to negligible levels and mRNA₂ became expressed and increased in abundance to account for 20% of the mRNA pool by the 15^th postnatal day. To examine the effect of T₃ on this developmental program, neonates were injected with 75 g T₃/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T₃ treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T₃ treatment, the abundances of mRNA₁, mRNA₃, and mRNA₁ increased, while mRNA₂ continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T₃ augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T₃ does not act as a molecular switch in the developmental expression of the mRNA isoforms in rat myocardium during the first four postnatal days.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length

FT-IR spectroscopy has been used to evaluate the acyl chain conformational ordering of DMPC, DMPE, DMPA (pH 6 and 12), DMPG (pH 1 and 7), and DPPC, DPPE, DPPA (pH 6). The frequencies of the symmetric and antisymmetric methylene stretching vibrations were determined as a function of temperature. In the liquid-crystalline phase the frequencies show a qualitative dependence on the amount of chain disorder. Quantitative data for trans-gauche isomerization were obtained from the integral intensities of the conformation sensitive methylene wagging absorptions at ca. 1368 cm^–1 (gtg and gtg sequences), 1356 cm ^–1 (double gauche) and 1342 cm^–1 (end gauche). The integral band intensities were converted to the number of gauche conformers per acyl chain using the calibration factors published by Senak et al. (1991). At 69°C the highest number of gauche conformers excluding contributions from single gauche conformers and jogs (gtttg) are found for PCs (DMPC: 2.6; DPPC: 2.4), followed by DMPG^– (2.0), phosphatidylethanolamines (DMPE: 1.4; DPPE: 2.0), protonated DMPG (1.5), and phosphatidic acids (DPPA^–: 1.7; DMPA^–: 1.4, DMPA^2–: 1.7). From ²H-NMR measurements of perdeuterated samples of DMPC, DMPA, DPPC, and DPPA the quadrupolar splittings _Qi and the order parameter S _CDi of the CD₂-segments close to the chain ends could be determined whereas splittings in the plateau region of the chains could not be resolved. The quadrupolar splittings are affected by trans-gauche isomerization, long axis rotation, and restricted wobbling motions of the acyl chains. In the simplest assumption, the order parameter S_CD can be expressed as a product of a segmental order parameter S and a lhain order parameter S . For comparison of the different lipids we used average order parameters S_CD, obtained by averaging over all values, and S determined from the total number of gauche conformers per chain by FT-IR-spectroscopy, to calculate an empirical average chain order parameter S. The combination of ²H-NMR and FT-IR results allows the estimation of the relative extent of chain wobbling for the different lipid molecules. S is lowest for PCs (S 0.475) while PEs (S 0.51) and PAs (S0.52) show less chain wobbling.Abbreviations FT-IR Fourier transform infrared - ²H-NMR deuterium nuclear magnetic resonance - DMPC(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylcholine - DMPE(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylethanolamine - DMPA(–d₅₄) (perdeuterated) dimyristoyl-phosphatidic acid - DMPG dimyristoyl-phosphatidylglycerol - DPPC(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylcholine - DPPE(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylethanolamine - DPPA(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidic acid - gtg gauche ^±-trans-gauche^± - gtg gauche^±-trans-gauche^± - dg double gauche - eg end gauche Correspondence to: A. Blume     

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.     

Alpha-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes

An -N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426^T. The -N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50°C and for a week at 20°C. The enzyme can be stored under the sterile conditions for any length of time at 4°C, but it does not withstand freezing. The -N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl--N-acetyl-D-galactosaminide, the K _m is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.     

Flight of the honey bee VI: energetics of wind tunnel exhaustion flights at defined fuel content,speed adaptation and aerodynamics

To gain information on extended flight energetics, quasi-natural flight conditions imitating steady horizontal flight were set by combining the tetheredflight wind-tunnel method with the exhaustion-flight method. The bees were suspended from a two-component aerodynamic balance at different, near optimum body angle of attack and were allowed to choose their own speed: their body mass and body weight was determined before and after a flight; their speed, lift, wingbeat frequency and total flight time were measured throughout a flight. These values were used to determine thrust, resultant aerodynamic force (magnitude and tilting angle), Reynolds number, total flight distance and total flight impulse. Flights in which lift was body weight were mostly obtained. Bees, flown to complete exhausion, were refed with 5, 10, 15 or 20 l of a 1.28-mol·l^-1 glucose solution (energy content w=18.5, 37.0, 55.5 or 74.0 J) and again flown to complete exhaustion at an ambient temperature of 25±1.5°C by a flight of known duration such that the calculation of absolute and relative metabolic power was possible. Mean body mass after exhaustion was 76.49±3.52 mg. During long term flights of 7.47–31.30 min similar changes in flight velocity, lift, thrust, aerodynamic force, wingbeat frequency and tilting angle took place, independent of the volume of feeding solution. After increasing rapidly within 15 s a more or less steady phase of 60–80% of total flight time, showing only a slight decrease, was followed by a steeper, more irregular decrease, finally reaching 0 within 20–30 s. In steady phases lift was nearly equal to resultant aerodynamic force; tilting angle was 79.8±4.0°, thrust to lift radio did not vary, thrust was 18.0±7.4% of lift, lift was somewhat higher/equal/lower than body mass in 61.3%, 16.1%, 22.6% of all totally analysable flights (n=31). The following parameters were varied as functions of volume of feeding solution (5–20 l in steps of 5 l) and energy content. (18.5–74.0 J in steps of 18.5 J): total flight time, velocity, total flight distance, mean lift, thrust, mean resultant aerodynamic force, tilting angle, total flight impulse, wingbeat frequency, metabolic power and metabolic power related to body mass, the latter related to empty, full and mean (=100 mg) body mass. The following positive correlations were found: L=1.069·10^-9 f ^2.538; R=1.629·10^-9 f ^2.464; P _m=7.079·10^-8 f ^2.456; P _m=0.008v+0.008; P _m=18.996L+0.022; P _m=19.782R+0.021; P _m=82.143T+0.028; P _m=1.245·bm _f ^1.424 ; P _{mrel e}=6.471·bm _f ^1.040 ; =83.248+0.385. The following negative correlations were found: V=3.939–0.032; T=1.324·10^-4–0.038·10^-4. Statistically significant correlations were not found in T(f), L(), R(), f(), P _m(bm _e), P _{m rel e}(bm _e), P _{m rel f}(bm _e), P _{m rel f}(bm _f).Abbreviations A(m²) frontal area - bl(m) body length - bm(mg) body mass - c(mol·1^-1) glucose concentration of feeding solution - c _D (dimensionless) drag coefficient, related to A - D(N) drag - F _w(N) body weight - F _wp weight of paper fragment lost at flight start - f wingbeat frequency (s^-1) - g(=9.81 m·s^-2) gravitational acceleration - I(Ns)=R(t) dt total impulse of a flight - L(N) lift vertical sustaining force component - P _m(J·s^-1=W) metabolic power - P_{m ret} (W·g^-1) metabolic power, related to body mass - R(N) resultant aerodynamic force - Re v·bl·v ^-1 (dimensionless) Reynolds number, related to body length - s(m) v(t) dt virtual flight distance of a flight - s(km) total virtual flight distance - T (N) thrust horizontal force component of horizontal flight - T _a (°C) ambient temperature - t(s) time - t _tot (s or min) total flight time - v(m·s^-1) flight velocity - v(l) volume of feeding solution - W (J) energy and energy content of V - ( °) body angle of attack between body longitudinal axis and flow direction - ( °) tilting angle ( 90°) between R and the horizont in horizontal flight v(=1.53·10^-5m²·s^-1 for air at 25°) kinematic viscosity - (=1.2 kg·m^-3 at 25°C) air density     

The responsiveness of Avena fatua aleurone protoplasts to gibberellic acid

The sensitivity to gibberellic acid (GA₃) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl₂ at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10^–7 M GA₃ than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA₃ and the production of -amylase. The dose response of aleurone protoplasts to GA₃, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10^–14 M GA₃. With increasing concentrations of GA₃, up to 10^–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA₃ is applied. GA₃-induced -amylase production increases markedly as the concentration is raised from 10^–9 M to 10^–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA₃. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA₃ control, than those from seed stored at 4°C, <35-fold higher than the minus GA₃ control. This apparent difference in the responsiveness of aleurone protoplasts to GA₃ could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Amylase activity and growth in internodes of deepwater rice

Isoelectrofocusing, product analysis, thermal denaturation studies and affinity chromatography on cycloheptaamylose-Sephadex were used to identify the amylolytic enzymes in internodes of deepwater rice (Oryza sativa L.). Amylolytic activity in internodes of deepwater rice consists of -amylase (sometimes separated into two isoforms) and of -amylase. During submergence of whole plants, -amylase activity increases in young, growing internodes, but -amylase activity declines. Although non-growing, mature internodes contain higher levels of -amylase than do the elongating younger internodes, the effect of submergence on amylase activities in both tissues follows the same trend. Submergence, gibberellic acid (GA₃) and ethylene all promote -amylase activity in growing and non-growing internodes of excised deepwater-rice stem sections. Inhibitor studies showed that submergence and ethylene promote -amylase activity in the absence of endogenous gibberellin (GA), and GA₃ enhances -amylase activity when ethylene action is inhibited. Therefore, ethylene and GA appear to increase -amylase activity independently of each other. Enhanced -amylase activities are probably responsible for the mobilization of carbohydrates which are needed to support internode elongation during submergence of deepwater rice.Abbreviations CHA cycloheptaamylose - GA₃ gibberellic acid - NBD 2,5-norbornadiene - TCY tetcyclacis