Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.     

Cross-linking study of the quaternary fine structure of mitochondrial F1-ATPase

A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation.     

pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

The D-glucose transporter is tissue-specific. Skeletal muscle and adipose tissue have a unique form of glucose transporter

Using isotopic equilibration with [3H]D-glucose and measurement of D-glucose inhibitable cytochalasin B binding, I show that the erythrocytes of embryonic and newborn rats contain D-glucose transporters. On the basis of cytochalasin B binding and the time course of isotopic exchange, the number of transporters in rat embryonic erythrocytes is only 5% of that in human erythrocytes. Antibodies raised against the human erythrocyte glucose transporter were used as a probe to investigate the structural similarity between transporters. On this basis, the polypeptides of the glucose transporter of human erythrocytes and of embryonic rat erythrocytes are similar but not identical; in addition, certain antibodies showed similar reactivity toward the transporter of rat embryonic erythrocytes and that of rat brain. These antibodies, however, react with brain transporters 5 to 10 times better than with those of skeletal muscle and adipocytes suggesting that insulin responsive tissues may have a different type of glucose transporter. The cellular location of glucose transporters in skeletal muscle, determined by immunofluorescence, is on the plasma membrane or very close to the plasma membrane.     

Effect of phosphorylation by cyclic AMP-dependent protein kinase on the smooth muscle actomyosin Mg2+-ATPase stimulatory activity of fodrin

Fodrin, a non-erythrocyte spectrin-like protein, has been purified from bovine brain and found to be phosphorylated by the cyclic AMP-dependent protein kinase with a maximal stoichiometry of 1.02 +/- 0.06 mol of phosphate/mol of fodrin dimer (n = 4). This phosphorylation was not affected by the presence of actin and calmodulin. The phosphorylation of fodrin was found to occur exclusively at serine residues on the beta subunit. Two-dimensional thin layer electrophoresis and chromatography of a tryptic digest of phosphorylated fodrin showed one major phosphopeptide and a few minor ones. We have previously reported that nonphosphorylated fodrin is capable of stimulating the smooth muscle actomyosin Mg2+-ATPase by 50-70% under a well-defined set of conditions such as a critical fodrin concentration and an optimal preincubation time (Wang, C., Ngai, P.K., Walsh, M.P., and Wang, J.H. (1987) Biochemistry 24, 1110-1117). We now report that phosphorylation of fodrin completely eliminates this stimulatory effect. However, phosphorylation of fodrin was able to compete with nonphosphorylated fodrin to result in the abolition of the stimulatory effect. Similarly, nonphosphorylated fodrin could overcome the inhibitory effect created by phosphorylated fodrin. The present results support the suggestion that the stimulation of the smooth muscle actomyosin Mg2+-ATPase by fodrin may be a physiological phenomenon and cyclic AMP may serve as a regulator for this effect.     

Fused cells of frog proximal tubule: I. Basic membrane properties

Summary Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca²⁺ and voltage activated, has a conductance of 221±7 pS, and can be inhibited by 10mm tetraethylammonium and by 1mm quinidine, but not by 4-aminopyridine, nor by 1mm Ba²⁺ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1±3.9 nSvs. a leak conductance of 1.01±0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5±1.4 pF.     

Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase

The gene gyrA of Escherichia coli, which encodes the A subunit of DNA gyrase (topoisomerase II), has been cloned and a region of approximately 3300 base-pairs sequenced. An open reading frame of 2625 nucleotides coding for a protein of 97,000 Mr is located. The peptide weight of the subunit predicted from this open reading frame is in close agreement with previously published estimates of that of the A subunit. There is a "TATAAT" promoter motif located 44 bases upstream from the first "ATG" of the open reading frame. The amino acid sequence derived from the nucleotide sequence is about 50% homologous with that derived from the Bacillus subtilis gyrA gene sequence, with several regions showing greater than 90% homology.     

Influence of microgravity on cellular differentiation in root caps of Zea mays

We launched imbibed seeds of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on cellular differentiation in root caps. The influence of microgravity varied with different stages of cellular differentiation. Overall, microgravity tended to 1) increase relative volumes of hyaloplasm and lipid bodies, 2) decrease the relative volumes of plastids, mitochondria, dictyosomes, and the vacuome, and 3) exert no influence on the relative volume of nuclei in cells comprising the root cap. The reduced allocation of dictyosomal volume in peripheral cells of flight-grown seedlings correlated positively with their secretion of significantly less mucilage than peripheral cells of Earth-grown seedlings. These results indicate that 1) microgravity alters the patterns of cellular differentiation and structures of all cell types comprising the root cap, and 2) the influence of microgravity on cellular differentiation in root caps of Zea mays is organelle specific.     

兜兰胚胎学的研究

云南兜兰花药壁有五层细胞,绒毡层细胞具双核,属分泌型。小孢子母细胞减数分裂为同时型。四分体中四个小孢子呈四面体或左右对称式排列。成熟花粉为二细胞型。单粒分散的花粉包裹于黄色粘性物质中。生殖细胞最初形成的壁为胼胝质的,待游离到营养细胞质中时,具一层很薄的 PAS 正反应的壁,直到花药开裂时这层壁仍存在。成熟花粉无特化的萌发孔,只具薄壁区。胚珠为薄珠心,具一层珠被,胚囊发育为葱型,成熟胚囊为6—8核,胚发育过程中,具2—4细胞胚柄。胚乳具二核。种子成熟时胚柄及胚乳核都消失。成熟种子只具单层细胞的种皮和一个未分化的球形胚。     

三尖杉属受精作用的研究

三尖杉属的精原细胞有一类似银杏生毛体的星状体结构,它分裂产生的2个精子,在大小与形态上都基本相同,而且在精子细胞质中具有拟核仁颗粒存在。上述结构是三尖杉属的重要特点之一。本属植物的成熟卵细胞特别长,细胞质中有丰富的拟核仁结构,卵核下方具2—3团浓稠的细胞质团,这些结构很像穗花杉的卵细胞。三尖杉属的受精作用,属于有丝分裂后类型,这种类型只在松科和三尖杉科中发现。受精后,卵细胞发生强烈的极性分化,上部细胞质变成高度液泡化;相反,下部细胞质则聚集大量蛋白泡和拟核仁颗粒。