The 59-kDa polypeptide constituent of 8–10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase

The fungus Neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kDa polypeptide (P59Nc). We have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (PDC, EC 4.1.1.1), which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. Immunofluorescence analyses of 8–10-nm filaments, as well as quantitative Northern blot studies of P59Nc mRNA and measurements of PDC activity, showed that the presence and abundance of PDC filaments depends on the metabolic growth conditions of the cells. These findings may be of relevance to the biology of ethanol production by fungi, and may shed light on the nature and variable presence of filament bundles described in fungal cells.     

菌紫质的Ser和Lys残基修饰对BR结构和功能的影响

用化学修饰研究了菌紫质（ＢＲ）的结构和功能的变化。用氮氧自由基分别对赖氨酸和丝氨酸进行修饰,研究结果表明在圆二色谱上（ＣＤ谱）,与天然紫膜样品比较,两种自由基分别修饰赖氨酸（Ｌｙｓ）和丝氨酸（Ｓｅｒ）残基２４小时后的ＣＤ谱中均只有负峰,分别在５９６ｎｍ和６０２ｎｍ,５３５ｎｍ的正峰已消失,７２小时后５３５ｎｍ的正峰部分地恢复,但１２０小时后均未见进一步恢复。与未修饰的紫膜相比,两种自由基修饰的紫膜在Ｒａｍａｎ光谱上观察到中间体Ｍ４１２的相对量要明显增加。本文对这二种化学修饰引起的ＢＲ结构和功能变化进行了初步讨论。     

Intraspecific variation for CO2 compensation point and differential growth among variants in a C3−C4 intermediate plant

CO₂ compenstation point (), the concentration of CO₂ at which photosynthesis and respiration are at equilibrium, is a commonly used diagnostic for the C₄ photosynthetic pathway, since it reflects the reduced photorespiration that is a property of C₄ photosynthesis. Geographic variation for was examined within Flaveria linearis, a C₃–C₄ intermediate species. Collections from four widely separated Floridian populations were propagated in a greenhouse and measured for . Little differentiation among populations was found, but significant within-population variation was present. Temperature is a hypothesized selective agent for the C₄ photosynthetic pathway. To test this hypothesis, plants exhibiting a range of were cloned and placed in growth chambers at 25°C and 40°C. After 7 weeks, valves were remeasured and plants were harvested and weighed. There was a poor correlation between initial and final measures of for a given genotype (r=0.38, P>0.1). Broad sense heritability for was computed to be 0.10. At 25°C, there was no relationship between final size and . At 40°C, more C₄-like plants, as indicated by their low , had grown larger. Differences in relative growth rate were attributable more to differences in net assimilation rate than in leaf area ratio. Taken together, these results demonstrate that although significant plasticity exists in the amount of photorespiration in this C₃–C₄ species, high temperature appears to be an effective selective agent for the reduction of photorespiration and the enhancement of C₄-like traits.     

Trypsin complexed with α1-proteinase inhibitor has an increased structural flexibility

Mutant rat trypsin Asp¹⁸⁹Ser was prepared and complexed with highly purified human α₁-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with α₁-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg¹¹⁷-Val¹¹⁸ one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.     

Identification of Ser-Pro and Thr-Pro Phosphorylation Sites in Chicken Neurofilament-M Tail Domain

Abstract: The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [³²P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. ³²P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.     

Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.