BATMAS30: amino acid substitution matrix for alignment of bacterial transporters

Aligned amino acid sequences of three functionally independent samples of transmembrane (TM) transport proteins have been analyzed. The concept of TM-kernel is proposed as the most probable transmembrane region of a sequence. The average amino acid composition of TM-kernels differs from the published amino acid composition of transmembrane segments. TM-kernels contain more alanines, glycines, and less polar, charged, and aromatic residues in contrast to non-TM-proteins. There are also differences between TM-kernels of bacterial and eukaryotic proteins. We have constructed amino acid substitution matrices for bacterial TM-kernels, named the BATMAS (BActerial Transmembrane MAtrix of Substitutions) series. In TM-kernels, polar and charged residues, as well as proline and tyrosine, are highly conserved, whereas there are more substitutions within the group of hydrophobic residues, in contrast to non-TM-proteins that have fewer, relatively more conserved, hydrophobic residues. These results demonstrate that alignment of transmembrane proteins should be based on at least two amino acid substitution matrices, one for loops (e.g., the BLOSUM series) and one for TM-segments (the BATMAS series), and the choice of the TM-matrix should be different for eukaryotic and bacterial proteins.     

植物氨基酸转运子研究进展

氨基酸是高等植物氮素同化产物长距离运输及在组织间分配的主要形式,通过跨膜转运的方式在植物体内进行运输。氨基酸转运子是位于生物膜上吸收及转运氨基酸的蛋白家族,对植物氮素营养具有重要贡献。本文对植物氨基酸转运子的表达、调控及其与氮素利用效率、植物产量与品质形成、抗逆性及适应性等方面的研究进展进行了综述。     

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.     

Effects of magnesium availability on the activity of plasma membrane ion transporters and light-induced responses from broad bean leaf mesophyll

Considering the physiological significance of Mg homeostasis in plants, surprisingly little is known about the molecular and ionic mechanisms mediating Mg transport across the plasma membrane and the impact of Mg availability on transport processes at the plasmalemma. In this study, a non-invasive ion-selective microelectrode technique (MIFE) was used to characterize the effects of Mg availability on the activity of plasma membrane H⁺, K⁺, Ca²⁺, and Mg²⁺ transporters in the mesophyll cells of broad bean (Vicia faba L.) plants. Based on the stoichiometry of ion-flux changes and results of pharmacological experiments, we suggest that at least two mechanisms are involved in Mg²⁺ uptake across the plasma membrane of bean mesophyll cells. One of them is a non-selective cation channel, also permeable to K⁺ and Ca²⁺. The other mechanism, operating at concentrations below 30 M, was speculated to be an H⁺/Mg⁺ exchanger. Experiments performed on leaves grown at different levels of Mg availability (from deficient to excessive) showed that Mg availability has a significant impact on the activity of plasma-membrane transporters for Ca²⁺, K⁺, and H⁺. We discuss the physiological significance of Mg-induced changes in leaf electrophysiological responses to light and the ionic mechanisms underlying this process.     

Complete amino acid sequence of jack bean urease

The subunit structure of jack bean urease has been unresolved in spite of many investigations. Thus far, the molecular weight for the native urease seem to range from 480,000 to 590,000 and the values for the monomer range from 30,000 to 97,000. The complete amino acid sequence of jack bean urease has been determined primarily by sequencing cyanogen bromide peptides, which were aligned by overlapping peptides obtained by lysylendopeptidase digestion of the protein and tryptic digestion of the citraconylated protein. The protein contains 840 amino acid residues in a single polypeptide chain and the subunit molecular weight calculated from the sequence is 90,790. The value of 544,740 for the hexamer, consistent with the value of 580,000 determined for intact urease by centrifugal analyses, indicated that urease consists of six subunits. Thirteen of 25 histidine residues in the urease subunit are crowded in the region between residues 479 and 607. Urease is a nickel metalloenzyme and the nickel has an essential role in catalysis by this enzyme. It is noteworthy that cysteine-592, which is recognized as essential for enzymatic activity and is related to the nickel ion in the active center, is located on this histidine-rich sequence.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Grouping of amino acid types and extraction of amino acid properties from multiple sequence alignments using variance maximization

Understanding of amino acid type co-occurrence in trusted multiple sequence alignments is a prerequisite for improved sequence alignment and remote homology detection algorithms. Two objective approaches were used to investigate co-occurrence, both based on variance maximization of the weighted residue frequencies in columns taken from a large alignment database. The first approach discretely grouped amino acid types, and the second approach extracted orthogonal properties of amino acids using principal components analysis. The grouping results corresponded to amino acid physical properties such as side chain hydrophobicity, size, or backbone flexibility, and an optimal arrangement of approximately eight groups was observed. However, interpretation of the orthogonal properties was more complex. Although the principal components accounting for the largest variances exhibited modest correlations with hydrophobicity and conservation of glycine, in general principal components did not correspond to physical properties of amino acids. Although not intuitive, these amino acid mathematical properties were demonstrated to be robust and to improve local pairwise alignment accuracy, relative to 20 amino acid frequencies alone, for a simple test case.     

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4

Summary. By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2. Received September 8, 2000 Accepted January 8, 2001     

Metabolite transport across the peribacteroid membrane during broad bean development

A temporal pattern of the peribacteroid membrane (PBM) transport function was studied. Spectrophotometric recording was used for establishing the effect of carbon-and nitrogen-containing substrates (malate, succinate, and glutamate) on the acidification of the peribacteroid space and the intensity of light scattering in the symbiosome suspension from broad bean (Vicia faba L.) root nodules of different age. At the early stages of nodule formation and functioning, PBM is permeable not only for malate and succinate, but also for glutamate, and this permeability fully provides for the active bacteroid division and the nitrogenase complex synthesis in the bacteroids at the expense of the carbon-and nitrogen-containing substrates. Mature nodules are characterized by the greatest nitrogen-fixing activity. In these nodules, PBM is selectively permeable for malate and succinate, but constitutes a barrier for glutamate. Thereby, mutually beneficial relations between the symbiotic partners are achieved. In senescent nodules, a rearrangement of symbiotic interactions is directed toward a minimization of both carbon and nitrogen metabolite consumption by the bacteroids. It is concluded that, in the course of the development of the legume-rhizobia symbiosis, the PBM transport function is changed. This function determines a qualitatively different pattern of symbiotic partner interactions in the following sequence: parasitism-mutualism-commensalism.     

SLC38A9: A lysosomal amino acid transporter at the core of the amino acid-sensing machinery that controls MTORC1

The mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) acts as a crucial regulator of cellular metabolism by integrating growth factor presence, energy and nutrient availability to coordinate anabolic and catabolic processes, and controls cell growth and proliferation. Amino acids are critical for MTORC1 activation, but the molecular mechanisms involved in sensing their presence are just beginning to be understood. We recently reported that the previously uncharacterized amino acid transporter SLC38A9 is a member of the lysosomal sensing machinery that signals amino acid availability to MTORC1. SLC38A9 is the first component of this complex shown to physically engage amino acids, suggesting a role at the core of the amino acid-sensing mechanism.     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

Two groups of thermophilic amino acid aminotransferases exhibiting broad substrate specificities for the synthesis of phenylglycine derivatives

Thirty two thermophilic amino acid aminotransferases (AATs) were expressed in Escherichia coli as soluble and active proteins. Based on their primary structures, the 32 AATs were divided into four phylogenetic groups (classes I, II, IV, and V). The substrate specificities of these AATs were examined, and 12 AATs were found capable of synthesizing ring-substituted phenylglycine derivatives such as hydroxyl-, methoxy-, and fluorophenylglycines. Eleven out of the 12 AATs were enzymes belonging to two phylogenetic groups namely, one subgroup of the class I family and the class IV family. AATs in these two groups may thus be useful for the synthesis of a variety of ring-substituted phenylglycine derivatives.     

Sheep twin chimaeras with admixtures of red cell amino acid and potassium transport phenotypes

A pair of sheep twins each had two populations of red cells. Population 1 was positive for antigens Aa, Ma and Mb, was low-potassium type, possessed an amino acid transport system and was lysine-negative phenotype. Population 2 was negative for antigens Aa, and Mb, was high-potassium type, lacked the amino acid transport system and was lysine-positive phenotype. Population 2 disappeared from both sheep over a period of 8 years.     

Different effects of hypothermia on amino acid incorporation and on amino acid uptake in the brainin vivo

The temperature dependence of the incorporation of amino acids into cerebral proteins and that of the transport of amino acids through the blood-brain barrier were studied. We measured the protein synthesis rate in vivo over a wide temperature range (14°C–38°C) in male Sprague-Dawley rats using a flooding dose of labeled valine. There was a linear dependence of the protein synthesis rate on temperature. The temperature quotient expressed as per cent decrease per 1°C was somewhat lower at the lower temperatures, a decrease from 7.8% in the 37.7–32.5°C range to 6.7% in the 25.5–14°C range. The transport of the three amino acids phenylalanine, lysine, and alanine, representing there transport systems, through the blood-brain barrier showed no temperature dependence in vivo. The results show that in hypothermia cerebral metabolic rates are lowered to a great extent, while some aspects of metabolic transport are not affected.     

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

源于解淀粉芽胞杆菌酚酸脱羧酶的克隆与表达

【背景】酚酸脱羧酶催化分解酚酸产生的4-乙烯基酚类物质可用于食品添加剂及香精香料行业,而酚酸脱羧酶的表达水平相对较低,因此,高水平的酚酸脱羧酶是工业规模生产4-乙烯基酚类物质的先决条件。【目的】克隆解淀粉芽胞杆菌的酚酸脱羧酶基因,实现在大肠杆菌中的高效异源表达,分析酚酸脱羧酶的底物特异性,并对其表达条件进行优化。【方法】通过PCR技术获得酚酸脱羧酶的基因,构建重组基因工程菌,将测序结果与其他酚酸脱羧酶序列进行比对,利用IPTG诱导方法高效表达蛋白。将重组酚酸脱羧酶与4种不同的底物进行反应,设计响应面试验对诱导条件进行优化。【结果】酚酸脱羧酶对对香豆酸、阿魏酸、咖啡酸、芥子酸的比酶活比率为:100:23.33:15.39:10.51。结合与其他酚酸脱羧酶比对结果发现酚酸脱羧酶家族的C末端区域氨基酸序列的变异率最高,这与酚酸脱羧酶的底物特异性和催化机制有关。通过单因素和响应面试验得到酚酸脱羧酶诱导表达的最佳条件为:2×YT培养基,诱导温度30°C,接种量1.78%,诱导时机3.8 h,IPTG1.25mmol/L,诱导时间18h,此时预测酶活和实际酶活分别为47.61IU/mL和47.55...     

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.