Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.     

Small sequence insertions within the branch point region dictate alternative sites of lariat formation in a yeast intron.

The problem of intron recognition in S. cerevisiae appears to be in part solved by the strong conservation of intron encoded splicing signals, in particular the 5' GUAUGU and the branch point UACUAAC which interact via base pairing with the RNA components of U1 and U2 snRNPs respectively. Nevertheless, the mere presence of such signals is insufficient for splicing to occur. In the S. cerevisiae ACT1 intron, a silent UACUAAC-like sequence (UACUAAG) is located 7 nucleotides upstream of the canonical branch point signal. In order to investigate whether other factors, in addition to the U2-UACUAAC base-pair interactions, affect branch point selection in yeast, we created a cis-competition assay by converting the UACUAAG to a strong branch point signal (UACUAAC). If simply having a canonical UACUAAC sequence were sufficient for lariat formation, a 1:1 ratio in usage of the two signals should have been observed. In this double branch point intron, however, the downstream UACUAAC is utilized preferentially (4:1). Results obtained from the analyses of numerous sequence variants flanking the two UACUAAC sequences, demonstrate that non-conserved sequences in the branch point region are able to define lariat formation. Consequently, we conclude that U2 base-pairing is not the only requirement determining branch point selection in yeast, and local structure in the vicinity of the branch point could play a critical role in its recognition.     

Products of the lipoxygenase pathway in human natural killer cell cytotoxicity

As earlier data suggested the importance of lipoxygenase activation for expression of human NK cell cytotoxicity, four different lipoxygenase inhibitors were tested for suppression of natural killer (NK) cell lysis. All inhibitors were found active at nontoxic concentrations with 50% inhibition at approximately 15 microM for nordihydroguaiaretic acid (NDGA). NK cell lysis could be reconstituted to NDGA-suppressed cells with leukotriene B4 (LTB4), the all-trans isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4, and 20-COOH-LTB4. LTB4 reconstitution was best in the concentration range 1-100 pM and near control levels at both higher and lower concentrations. Herpesvirus Ateles-transformed killer T cells could also be inhibited by NDGA. These data indicate that lipoxygenase activity is required for human NK cell lysis and that several different LTB4-related products can restore NK activity in inhibited cells; they also suggest that the lipoxygenase pathway is present in the killer cell population.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

Construction of a cosmid library of Spirulina platensis as an approach to DNA physical mapping

Abstract A Spirulina platensis gene library has been constructed using cosmid vector pMMB34. The cosmid bank was controlled for its random gene distribution by colony hybridization. Genes were identified using either homologous or heterologous probes of genes involved in photosynthesis (large and small subunit of d -ribulose 1,5-bisphosphate carboxylase, 32 kDa thylakoid protein, α, β subunits of C-phycocyanin) and protein synthesis (elongation factors EF-Tu, EF-G).     

Respiratory mechanics in mechanically ventilated patients with respiratory failure

In 11 mechanically ventilated patients, respiratory mechanics were measured 1) during constant flow inflation and 2) following end-inflation airway occlusion, as proposed in model analysis (J. Appl. Physiol. 58: 1840-1848, 1985. During the latter part of inflation, the relationship between driving pressure and lung volume change was linear, allowing determination of static respiratory elastance (Ers) and resistance (RT). The latter represents in each patient the maximum resistance value that can obtain with the prevailing time constant inhomogeneity. Following occlusion, Ers and RT were also obtained along with RT (min) which represents a minimum, i.e., resistance value that would obtain in the absence of time constant inhomogeneity. A discrepancy between inflation and occlusion Ers and RT was found only in the three patients without positive end-expiratory pressure, and could be attributed to recruitment of lung units during inflation. In all instances Ers and RT were higher than normal. RT(min) was lower in all patients than the corresponding values of RT, indicating that resistance was frequency dependent due to time constant inequalities. Changes in inflation rate did not affect Ers, while RT increased with increasing flow.