Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacteriumSulfolobus solfataricus

ASulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (G1–G6) and one of them (G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of theS. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.This work was supported by grants from the CNR, Target Project on Biotechnology and Bioinstrumentation, and MURST (Rome).     

Allosteric regulation of NAD(NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase from Chlorella by α-amino acids,dithiothreitol and ATP

α-Amino acids (glycine, serine, histidine, aspartic acid and cysteine) and dithiothreitol (DTT) have been shown to activate both activities of the NAD(NADP)-dependent glyceraldehyde-3-phosphate dehydrogenase from Chlorella. The activation is allosteric and reaches 200–700%. The Hill coefficient values are close to 2 with all activators. ATP activates NADP-dependent but inhibits NAD-dependent activity, n_app and K values being the same for both enzyme activities. In this case positive cooperativity is also observed (n_app = 2.2). The present findings reveal the possible regulation of GAPD function in Chlorella with each of the coenzymes.     

Variation in the physiological sensitivity of maize inbreds to EMS a possible correlation with alcohol dehydrogenase activity

Three inbred lines of maize, AD-1, AD-7 and AD-19, showed significant differences in their physiological sensitivity to ethyl methanesulfonate (EMS). Among the seedling parameters tested, the width of the leaves was the most sensitive and showed the best correlation with the results obtained at maturity. Plant weight was also a good seedling parameter. The physiological sensitivity to EMS was negatively correlated with alcohol dehydrogenase (ADH) activity in the scutellum of the three lines. A possible explanation for this correlation is discussed; however, it may have been due to chance.     

Changes in enzyme activity of white yam tubers after prolonged storage

There is a substantial increase in the activities of phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in white yam tubers as they age. The high glucose-6-phosphate dehydrogenase activities suggest that the pentose phosphate pathway is important in yam tuber tissue.     

The effect of training and detraining on lactate dehydrogenase isoenzymes in the horse

In the horse, total LDH activity increased with training and the H and M subunit activity parallelled this increase. It is suggested that these increases are in response to a stimulus from the type of training program utilised. The first half of a detraining program decreased the activity of the H and M subunits as might be expected. A sharp rise in the total LDH and the M subunit activity occurred during the latter half of the detraining program. This unexpected increase may be due to relatively more hypoxic conditions prevailing in the muscle during the detraining period.     

Analysis of genetic variation affecting the relative activities of fast and slow ADH dimers in Drosophila melanogaster heterozygotes

When Adh ^F /Adh ^S heterozygote homogenates are stained after electrophoresis, considerable variation is observed in the activity ratio of the FF dimer to the SS dimer. Two Adh ^S strains showed a sharp, consistent difference when crossed to a common Adh ^F strain. Optical scanning and genetic analysis confirmed that this difference originates close to the Adh locus. Since the morphs varied concordantly in their activities on numerous alcohols, and since aging and heat-treatment experiments failed to reveal a stability difference, it is proposed that the difference is regulatory in nature, affecting ADH synthesis and primarily cis-acting. A survey of wild flies revealed additional variation in the FF/SS activity ratio. Further genetic analysis showed that the basis of this variation is not restricted to the second chromosome. Furthermore, modification of the activity ratio implies some degree of allelespecificity on the part of the modifiers.This work was supported in part by money collected by Jewish Community of Iowa City, Iowa, and by NSF Grant 76-01903 to Roger Milkman.     

Effect of 2,3-dinitrilo-1,4-dithia-9,10-antraquinone on Ehrlich ascites carcinoma and yeast cells

The inhibition of glycolysis by 2,3-dinitrilo-1,4-dithia-9,10-antraquinone (DDA) in Ehrlich ascites carcinoma (EAC) cells as well as in the investigated respiratory and fermentative strains of yeasts was found to be the result of inactivation of thiol enzymes of this pathway. Increasing concentration of DDA caused, in EAC cells, marked inhibition of hexokinase (HK), phosphofructokinase (PFK) and practically total inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These three enzymes, as well as alcohol dehydrogenase (ADH) were also inactivated by DDA in yeasts. DDA inhibited the biosynthetic processes as measured by following the rate of [14C]adenine and [14C)]valine incorporation into TCA-precipitable fractions proportionally to the degree of glucose consumption by EAC or the yeast cells.     

Activity of some glycolytic and pentose phosphate pathway enzymes during the development of adventitious roots

Activities of phosphofructokinase (PFK, EC 2.7.1.11), glyceraldehyde 3-phosphate (NAD) dehydrogenase [G-3-PD(NAD), EC 1.2.1.12], glucose 6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44) were determined in bean cuttings (Phaseolus vulgaris L. cv. Top Crop) over 4 days, encompassing adventitious root primordium initiation and development. Effects of applied auxin and “endogenous root-forming stimulus”(ERS) on enzyme activities, concentrations of reducing sugars, and primordium development were also determined during the first 4 days of propagation. Effects of auxin were determined through use of applied indole-3-acetic acid (IAA) or 2,3,5-triiodobenzoic acid. Effects of ERS were evaluated by means of decapitation of cuttings. Increased basipetal transport and increased metabolism of reducing sugars occurred in leafy cuttings in response to applied IAA and to ERS. Primordium development and activities of the four enzymes increased in leafy cuttings under conditions that simultaneously increased basipetal transport and metabolism of reducing sugars. Three types of enzyme activity response were found: (i) activity increased over time by ERS and by applied IAA [G-3-PD(NAD)], (ii) activity increased over time by ERS but not by applied IAA (PFK, G-6-PD), (iii) activity increased over time but not by ERS or applied IAA (6-PGD). Increases in G-3-PD(NAD), G-6-PD, and PFK activity in leafy cuttings were positively related to primordium development. 6-PGD activity increased in leafy cuttings during primordium development and may have supported it. However, equal increases occurred in decapitated cuttings, in which the long-term development of primordia was supressed. Results for G-3-PD(NAD) that were obtained in an experiment with jack pine (Pinus banksiana Lamb.) seedling cuttings were similar to results for the same enzyme in bean cuttings. G-3-PD(NAD) activity in naphthaleneacetic acid-treated jack pine cuttings increased with time, in comparison with untreated cuttings, before root emergence.     

酿酒酵母ADH3基因的敲除

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。     

Diagnostic performance of lactate dehydrogenase (LDH) isoenzymes levels for the severity of COVID-19

BackgroundLactate dehydrogenase (LDH) levels predict coronavirus disease 2019 (COVID-19) severity. We investigated LDH isoenzyme levels to identify the tissue responsible for serum LDH elevation in patients with COVID-19.MethodsHospitalised COVID-19 patients with serum LDH levels exceeding the upper reference limit included. LDH isoenzymes were detected quantitatively on agarose gels. The radiological severity of lung involvement on computed tomography was scored as 0-5 for each lobe (total possible score, 0-25). Disease severity was determined using the World Health Organization (WHO) clinical progression scale.ResultsIn total, 111 patients (mean age, 59.96 ± 16.14), including 43 females (38.7%), were enrolled. The serum levels of total LDH and all five LDH isoenzymes were significantly higher in the severe group. The levels of all LDH isoenzymes excluding LDH5 positively correlated with the WHO score. LDH3 levels correlated with chest computed tomography findings (r² = 0.267, p = 0.005). On multivariate analysis, LDH3 was an independent risk factor for the deterioration of COVID-19.ConclusionsLDH3 appears to be an independent risk factor for deterioration in patients with COVID-19. LDH elevation in patients with COVID-19 predominantly resulted from lung, liver and muscle damage.     

Hypoxic induction of alcohol and lactate dehydrogenases in lupine seedlings

Seedlings of lupine (Lupinus luteus L. cv. Juno) were exposed for up to 96 hours to 1 to 2 kPa partial pressure oxygen (hypoxic treatment) and activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and their isoform profiles were determined. Roots of lupine seedlings were grown in a nitrogen flushed nutrient solution while their shoots were in air. Prolonged hypoxia led to a reduction of root elongation. This was accompanied by reduced increase in dry weight suggesting that insufficient carbohydrate supply was the cause of retarded growth of lupine roots. Hypoxically treated roots showed induction of ADH and LDH acivities. The maximum increase in LDH activity was low (2-fold) in contrast to ADH activity, which increased up to 7-fold. Hypoxic treatment of roots did not affect the activities of ADH and LDH in hypocotyls and cotyledons. Analysis of ADH and LDH activity gels indicated in roots 1 and 2 isoforms, respectively. The level of isozymes of both enzymes increased in roots upon exposure to hypoxic stress. Differences in isoenzymatic spectrum of ADH and LDH between roots, hypocotyls and cotyledons indicate organ specificity of isozymes of both enzymes. The importance of alcohol and lactate fermentation in roots to cope with hypoxic stress is discussed.     

Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii

The titers of key enzymes of xylose metabolism were measured and correlated with the kinetics of xylitol production by Debaryomyces hansenii under different oxygen transfer rates (OTR) in a batch reactor. An OTR change from 2.72 to 4.22 mmol O₂ l⁻¹ min⁻¹ resulted in a decrease in NADPH-dependent xylose reductase (XR) and NAD ± -dependent xylitol dehydrogenase (XDH) activities. For higher values of OTR (12.93 mmol O₂ l⁻¹ min⁻¹, the XDH titer increased twofold whereas the XR titer did not show a significant change. At the lowest OTR (2.72 mmol O₂ l⁻¹ min⁻¹), xylitol (and ethanol) production rates showed the highest values. However, xylitol specific productivity was twice as high as ethanol specific productivity. The titer of the NADPH-forming enzyme, glucose-6-phosphate dehydrogenase (GPDH), increased from 333 to 412 mU mg⁻¹ when the OTR was increased. However, 6-phosphogluconate dehydrogenase (PGDH) activity remained unchanged and at a lower level, which indicates that this enzyme is responsible for the carbon flux control of the oxidative branch of the pentose phosphate pathway. The activity of the alcohol-forming enzyme was repressed at the higher amount of oxygen, decreasing its activity more than 50%. The changes in ADH suggested that two different metabolic regions under oxygen-limited conditions can be hypothesized for xylose metabolism by D. hansenii. For low OTR values (up to 4.22 mmol O₂ l⁻¹ min⁻¹), a fermentative-type activity is displayed. At higher OTR values (above 4.22 mmol O₂ l⁻¹ min⁻¹), no significant fermentative activity is reported.     

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the T_m values of the heat-absorption curves decreased by 6.0 and 3.3 °C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD⁺-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35 M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311–H124 are significant for the enhanced stability of GAPDS. The salt bridge D311–H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.     

The structural basis of negative cooperativity: receptors and enzymes

Crystal structures of the negatively cooperative aspartate receptor caught at intermediate stages in the binding process help to elucidate structural factors involved in ligand binding. The frequency of occurrence of negatively cooperative proteins suggests that sequential changes in binding patterns will be extensive in positively cooperative as well as in negatively cooperative and no cooperativity proteins.