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81.
Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves
of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene
probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level
was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises
a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences
from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation
of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino
acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about
half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence
in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates
that the bi-specific NR is encoded by a single copy gene in birch.
These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097 相似文献
82.
V. Frankard M. Ghislain I. Negrutiu M. Jacobs 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,82(3):273-282
Summary Mutagenesis and the subsequent selection of mesophyll diploid protoplasts ofNicotiana sylvestris on growth inhibitory concentrations of lysine plus threonine has led to the isolation of an LT-resistant mutant. Regeneration of this line (RLT 70) and analysis of its descendants demonstrated the dominant monogenic nuclear character of the resistance gene, further namedak-LT1. When the inhibition properties of aspartate kinase were examined in the homozygous mutant, lysine-sensitive activity could no longer be detected. In comparison, 70%–80% of the wild-type enzyme activity was usually inhibited by lysine, and the rest by threonine. Evidence for the existence of at least two AK isoenzymes was obtained by ion-exchange chromatography, where two peaks of activity could be detected: the first one to be eluted is lysine sensitive, and the second one threonine sensitive. One consequence of the altered regulation of AK in the mutant was the enhanced production of soluble threonine. Threonine accumulation was observed to occur throughout the life cycle of the mutant plant as well as in its different organs. In particular, leaves exhibited a 45-fold increment of soluble threonine, which corresponds to a 13-fold increase in total threonine: almost one-third of the total amino acids was free and proteinbound threonine. In RLT 70 seeds, 20% of the free amino acid pool was in the form of threonine (70-fold accumulation compared to the wild type), and total threonine content was increased five fold. As a general rule, the other amino acids were also more abundant in RLT 70 seeds, such that the total of amino acids present was between two to four times higher, but in contrast with the situation encountered in leaves, this was also due to a higher protein-bound amino acid content. 相似文献
83.
Maria Ransjö 《Bioscience reports》1991,11(4):203-211
The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 mol/l) and choleratoxin (0.1 mg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1mol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase. 相似文献
84.
Morris F. White 《Journal of bioenergetics and biomembranes》1991,23(1):63-82
Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism. 相似文献
85.
人肌肌酸激酶胍变性时的失活与构象变化的比较研究 总被引:1,自引:1,他引:0
应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100%,则天然酶酪氨酸残基的暴露程度只有2%。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。 相似文献
86.
溴化十烷基三甲铵对肌酸激酶的变性及复性 总被引:6,自引:1,他引:5
测定了溴化十烷基三甲铵(C_(10)H_(21)N(CH_3)_3Br;记为C_(10)NM_3)对肌酸激酶(Creatine Kinase;记为C.K.)的活力及构象的影响,以及变性后C.K.的复性。实验结果表明:C_(10)NM_3对C.K.有很强的变性能力,在0.06M时,就可以使C.K.完全失活;在0.08M时,就可使C.K.内部的6个巯基有4个暴露出来;与SDS变性剂不同,C.K.在高浓度的C_(10)NM_3中变性以后,直接冲稀时就可以完全复性。FTIR以及CD等实验方法证明,尽管C_(10)NM_3能使C.K.的构象发生明显变化,但C.K.的二级结构几乎不受C_(10)NM_3的影响。 相似文献
87.
88.
A phosphorylated analogue of DSIP at Ser7 has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent Km and Vmax values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro. 相似文献
89.
The activating factor of ATP·Mg-dependent protein phosphatase (F
A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F
A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK
m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK
m value of about 3 µM. When using microtubules as substrates,F
A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, theF
A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF
A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF
A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF
A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F
A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules. 相似文献
90.
The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the -subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of 32P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the -subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn2+ (Ka = 1.0 mM) was much better than Mg2+ (Ka = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t1/2 = 3.6 min) proceeded at a much slower rate compared to that of the -subunit of IR (t1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4 : 1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR -subunit suggest that pp43 was not derived from IR -subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase. 相似文献