烧伤患者血浆和红细胞游离氨基酸变化及输注氨基酸对其变化的影响

动态测定烧伤患者血浆及红细胞内游离氨基酸的含量 ,探讨输入外源性氨基酸后对血及红细胞内游离氨基酸的影响。以日立 835— 5 0型氨基酸自动分析仪测定烧伤患者血浆及红细胞内游离氨基酸含量。结果发现烧伤患者血浆总游离氨基酸浓度从伤后到 2 1天均显著降低 (P <0 .0 5～ 0 .0 1) ;赖、苯丙和苯丙 酪氨酸比值显著升高 (P <0 .0 5～ 0 .0 1) ;色、组、精、丙、甘、苏、脯和丝氨酸比值显著降低 (P <0 .0 5～ 0 .0 1) ;缬、亮、异亮、酪、胱和支链氨基酸伤后早期降低。烧伤患者红细胞内总游离氨基酸含量不同程度降低 ,其中 1、3、7天降低显著 (P <0 .0 5～ 0 .0 1) ;红细胞内苯丙和苯丙 酪氨酸比值未见显著性升高 ;色、蛋、精、脯氨酸含量很低或基本未测出。输注复合氨基酸注射液后未能显著改善患者血及红细胞内游离氨基酸含量。结果提示烧伤患者红细胞内游离氨基酸含量的变化趋势与血浆游离氨基酸变化趋势基本一致 ;烧伤后红细胞内苯丙氨酸及苯丙 酪氨酸比值有别于血浆变化。本研究条件下补充外源性氨基酸未能显著改变烧伤患者血浆及红细胞内游离氨基酸的含量     

肝硬化与肝癌患者血浆游离氨基酸水平分析

采用高效液相色谱法分析了２５例肝硬化和１５例肝癌患者空腹血浆游离氨基酸水平的变化。结果表明,二者支链氨基酸（ＢＣＡＡ）如Ｖａｌ、Ｉｌｅ呈下降趋势,而芳香族氨基酸（ＡＡＡ）如Ｔｙｒ、Ｐｈｅ则呈上升趋势,ＢＣＡＡ／ＡＡＡ分子比值下降。丙氨酸（Ａｌａ）、蛋氨酸（Ｍｅｔ）、谷氨酰胺（Ｇｌｎ）及天门冬氨酸（Ａｓｐ）明显上升。其变化趋势二者存在差别。     

肾小球疾病儿童体内游离氨基酸含量的研究

为了研究小儿肾小球病变时体内游离氨基酸的代谢变化,采用丹酰氯聚酰胺薄层分析法研究了原发性肾病综合症（ＩＮＳ）８例、急性肾小球肾炎（ＡＧＮ）１２例、过敏性紫癜肾炎（ＡＰＮ）７例与正常对照组２９例的血清及红细胞内游离氨基酸含量的变化。结果表明：（１）此三种肾小球疾病,血清中苯丙氨酸（Ｐｈｅ）、脯氨酸（Ｐｒｏ）、色氨酸（Ｔａｐ）、赖氨酸（Ｌｙｓ）、甘氨酸（Ｇｌｙ）明显高于正常,导致酪／苯丙（Ｔｙｒ／Ｐｈｅ）、缬／甘（Ｖａｌ／Ｇｌｙ）等分子比降低,提示肾功能受损。（２）血清支链氨基酸（ＢＣＡＡ）的含量在ＩＮＳ中低于正常组（ｔ＝３．４８;Ｐ＜０．０１）,而在ＡＧＮ、ＡＰＮ中却高于正常组（ｔ分别为２．３３,２．３９,Ｐ＜０．０５）。（３）红细胞中丝氨酸（Ｓｒｅ）、苯丙氨酸（Ｐｈｅ）、色氨酸（Ｔｒｐ）、胱氨酸＋半胱氨酸（Ｃｙｓ）、天冬氨酸（Ａｓｐ）、谷氨酸（Ｇｌｕ）的含量ＡＰＮ组高于ＩＮＳ及ＡＧＮ,提示ＡＰＮ患儿中肾功能损害没有ＩＮＳ及ＡＧＮ严重。     

慢性肾炎体液氨基酸与正常比较

慢性肾炎体液氨基酸与正常比较蒋滢,黄美英,何达纯ＣｈａｎｇｅｏｆＦｌｕｉｄＦｒｅｅＡｍｉｎｏＡｃｉｄｉｎＰａｔｉｅｎｔｓＷｉｔｈＣｈｒｏｎｉｃＮｅｐｈｒｉｔｉｓ￥ＪｉａｎｇＹｉｎｇ;ＨｅＤａｃｈｕｎａｎｄＨｕａｎｇＭｅｉｙｉｎｇ（Ｄｅｐａｒｔｍｅｎｔ...     

The effect of N-stearoylethanolamine on free amino acid levels in plasma and liver of rats with an experimental burn

The effect of an endocannabinoid congener, N-stearoylethanolamine (NSE), on the content of plasma and liver pools of free amino acids (AA) was studied in burned rats. After application of a thermal skin burn (stage III) animals perorally received an aqueous suspension of NSE (10 mg/kg of body weight) during 7 days or were treated with the aqueous NSE suspension (10 mg/ml) applied onto the burn wound, or received a combined treatment. It has been originally demonstrated for the first time, that the treatment of burned rats with NSE prevented the decrease in total AA concentration in blood plasma and the increase in hepatic AA concentration due to modulation in concentrations of glycogenic AA. In burned animals the ratio of plasma and liver homogenate Phe/Tyr and Gly/Val increased while the Fischer ratio (Ile+Leu+Val/Phe+Tyr) decreased, and after the treatment with NSE these parameters remained at the level of intact animals. These data demonstrate that NSE possesses adaptogenic properties, and it is involved in the organism response to the burn. This prevents changes in blood plasma and hepatic pools of free AA of NSE-treated rats with the burn wound compared with untreated animals.     

辐射损伤对动物血浆游离氨基酸的影响

<正> 当前不同氨基酸比例配制的混合液已广泛应用于许多疾病的支持治疗,但混合氨基酸对辐射损伤患者有无支持疗法的作用,尚待研究。本文使用DNS—Cl(Dimet-hylamino—Naphthalene—5—Sulfonylchlovide,丹酰氯)荧光标记,聚酰胺(polyamide)薄膜层析方法,测定血浆中13种游离氨基酸,确定哪些氨基酸与辐射损伤有关,以此探讨(1)辐射损伤血浆氨基酸的敏感指标;(2)在此基础上,为配制不同氨基酸比例的混合液作为辐射损伤支持疗法,提供一定的科学依据。     

Tyrosine requirements in children with classical PKU determined by indicator amino acid oxidation

Tyrosine (Tyr) is an essential amino acid in phenylketonuria (PKU) because of the limited hydroxylation of phenylalanine (Phe) to Tyr. The recommended intakes for Tyr in PKU are at least five times the recommended phenylalanine intakes. This suggests that Phe and Tyr contribute approximately 20 and 80%, respectively, of the aromatic amino acid (AAA) requirement (REQ). In animals and normal humans, dietary Tyr was shown to spare 40-50% of the Phe requirement, proportions that reflect dietary and tissue protein composition. We tested the hypothesis that the Tyr REQ in PKU would account for 45% of the total AAA REQ by indicator amino acid oxidation (IAAO). Tyr REQ was determined in five children with PKU by examining the effect of varying dietary Tyr intake on lysine oxidation and the appearance of (13)CO(2) in breath (F(13)CO(2)) under dietary conditions of adequate energy, protein (1.5 g x kg(-1) x day(-1)), and phenylalanine (25 mg x kg(-1) x day(-1)). Lysine oxidation and F(13)CO(2) were determined using a primed 4-h oral equal-dose infusion of L-[1-(13)C]lysine. Lysine oxidation and F(13)CO(2) decreased linearly as Tyr intake increased, to a break point that was interpreted as the mean dietary Tyr requirement (16.3 and 19.2 mg x kg(-1) x day(-1), respectively). At Tyr intakes of >16.3 and 19.2 mg x kg(-1) x day(-1), lysine oxidation and F(13)CO(2), respectively, were low and constant. This represents 40.4 and 44.4%, respectively, of the total AAA intake. The current recommendations for Tyr intake in PKU patients appear to be overestimated by a factor of approximately 5. This study is the first application of the IAAO technique in a pediatric population and in humans with an inborn error of metabolism.     

A diurnal component to the variation in sieve tube amino acid content in wheat

We have used high-sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection method to quantify 16 amino acids in wheat (Triticum aestivum) sieve tube (ST) samples as small as 2 nL collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual STs. Some of the total concentrations of amino acids recorded are higher than those reported previously. The results obtained show variation in the concentrations of phenylalanine (Phe), histidine/valine (His/Val), leucine/isoleucine (Leu/Ile), arginine, asparagine, glutamine, tyrosine (Tyr), and lysine (Lys) across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (Tyr, Lys, Phe, His/Val, and Leu/Ile) that showed striking covariation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of Tyr, Lys, Phe, Leu/Ile, His/Val, asparagine, arginine, and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analyzing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed: samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.     

Characteristic of aromatic amino acid substitution at alpha 96 of hemoglobin

Replacement of valine by tryptophan or tyrosine at position alpha96 of the alpha chain (alpha96Val), located in the alpha(1)beta(2) subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the alpha96 position. The characteristic of aromatic amino acid substitution at the alpha96 of hemoglobin has been further investigated by producing double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp). r Hb (alpha42Tyr --> Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between alpha42Tyr and beta99Asp in thealpha(1)beta(2) subunit interface of deoxy Hb A. The second mutation, alpha96Val -->Trp, may compensate the functional defects of r Hb (alpha42Tyr --> Phe), if the stability due to the introduction of trypophan at the alpha 96 position is strong enough to overcome the defect of r Hb (alpha42Tyr --> Phe). Double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb (alpha42Tyr --> Phe). (1)H NMR spectroscopic data of r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between alpha 42Tyr and beta 99Asp is essential for the novel oxygen binding properties of deoxy Hb (alpha96Val --> Trp) .     

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --> Phe/Tyr324 --> Phe, factor VaFF; Glu330 --> Met/Val331 --> Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --> Phe/Tyr324 --> Phe and Glu330 --> Met/Val331 --> Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Conversion of amino acids into aroma compounds by cell-free extracts of Lactobacillus helveticus

AIMS: Lactobacillus helveticus is an essential starter in Swiss-type cheeses such as Emmental. This study was to determine whether cell-free extracts of Lact. helveticus were able to convert free amino acids into neutral volatile aroma compounds at the pH and temperature occurring in cheese. METHODS AND RESULTS: A mix of branched-chain (Leu, Ile, Val), aromatic (Tyr, Phe) and sulphur (Met) amino acids was incubated for 7 days, at pH 5.7 and 24 degrees C, with cell-free extracts of six strains. The amino acids were all transaminated into the corresponding keto acids when an amino group acceptor (alpha-ketoglutaric acid) was provided. Phe and Tyr were transaminated the most efficiently, followed by Leu, Met, Ile and Val. Three major volatile compounds were detected by GC-MS: benzaldehyde, dimethyl disulphide and 2-methyl propanol. Whatever the strain, benzaldehyde was produced in the highest quantity (0.25-1 micromol l(-1) mg(-1) protein). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus helveticus intracellular enzymes could significantly contribute to the production of aroma compounds from amino acid catabolism.     

Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts

Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.     

Phenylalanine and tyrosine kinetics in compensated liver cirrhosis: effects of meal ingestion

We explored the mechanism(s) of increased aromatic amino acids concentrations in liver cirrhosis using phenylalanine (Phe) and tyrosine (Tyr) isotope infusions in male patients with compensated cirrhosis (five in Child Class A, three in B) and in eight matched healthy controls, in both postabsorptive and fed states. After a baseline period, a standard liquid mixed meal was fed continuously over 4 h. Both a "plasma" and an intracellular model were employed. In the patients, steady-state Phe and Tyr concentrations were approximately 30-50% greater, and rates of Phe appearance (Ra) (plasma model), Tyr Ra, and Phe hydroxylation (Hy; both models) were approximately 25 to >100% greater than in controls in both states. Meal ingestion increased (P<0.05 or less vs. basal) Phe and Tyr concentrations, Phe and Tyr Ra, Phe Hy, and % Tyr Ra not deriving from Hy in both groups. Hy and Tyr Ra remained>50% greater (P<0.04 to P<0.01) in patients, whereas Phe Ra was more modestly increased. Phe utilization for protein synthesis increased similarly in both groups. Tyr clearance was normal, whereas Phe clearance tended to be lower (P=0.09, intracellular model) in the patients. In summary, in compensated liver cirrhosis studied under fasted and fed states, 1) Tyr Ra is increased; 2) Phe Hy and Phe Ra (plasma model) are increased; 3) Tyr clearance is normal; and 4) Phe clearance is slightly decreased. In conclusion, in cirrhosis increased total tyrosine Ra and hydroxylation contribute to fasting and postmeal hypertyrosinemia, whereas the mechanism(s) responsible for the hyperphenylalaninemia may include both increased production and decreased disposal.     

Amino acids defining the acyl pocket of an invertebrate cholinesterase

Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k_cat for BsCh; K_m and K_ss are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k_cat for BsCh and a decrease in k_cat for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by π–π or cation–π interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.     

Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.     

Functional consequences of engineering the hydrophobic pocket of carbonic anhydrase II.

Twelve amino acid substitutions of varying size and hydrophobicity were constructed at Val 143 in human carbonic anhydrase II (including Gly, Ser, Cys, Asn, Asp, Leu, Ile, His, Phe and Tyr) to examine the catalytic roles of the hydrophobic pocket in the active site of this enzyme. The CO2 hydrase and p-nitrophenyl acetate (PNPA) esterase activities, the pKa of the zinc-water ligand, the inhibition constant for cyanate (KOCN), and the binding constants for sulfonamide inhibitors were measured for various mutants and correlated with the size and hydrophobicity of the substituted amino acid. The kcat/KM for PNPA hydrolysis and KOCN are linearly dependent on the hydrophobicity of the amino acid at position 143. All of the activities of CAII are decreased by more than a factor of 10(3) when large amino acids (Phe and Tyr) are substituted for Val 143, but the CO2 hydrase activity is the most sensitive to the size and structure of the substituted amino acid. Addition of a single methyl group (V143I) decreases the activity 8-fold, while substitution of valine by tyrosine essentially destroys the enzyme function (kcat/KM for CO2 hydration is decreased by more than 10(5)-fold). KOCN does not increase until Phe is substituted for Val 143, suggesting that the cyanate and CO2 binding sites are not identical. The functional data in conjunction with X-ray crystallographic studies of four of the mutants [Alexander et al., 1991 (following paper in this issue)] allow interpretation of the mutants at a molecular level and mapping of the region of the active site important for CO2 association. The hydrophobic pocket, including residues Val 121 and Val 143, is important for CO2 and PNPA association; if the pocket is blocked, substrates cannot approach the zinc-hydroxide with the correct orientation to react. The interaction between Val 143 and CO2 is relatively weak (less than or equal to 0.5 kcal/mol) and nonspecific; the association site does not tightly hold CO2 in one fixed orientation for reaction with the zinc-hydroxide. This mechanism of catalysis may reflect a decreased requirement for specific orientation by CO2 since it is a symmetrical molecule.     

肾脏疾病患者体液氨基酸模式

使用 DNS— cl( Dimethylamino-naphthylane-5-sulfony chloride)荧光试剂 ,标记氨基酸成 DNS— AAs( DNS_ Amino Acids) ,然后采用聚酰胺薄膜层析 ,制成具有 2 0余种 DNS— AAs的荧光薄膜层析图谱 ,选择其中所需要检测的氨基酸 ,分别经洗脱后在日本岛津 RF— 51 0荧光分光光度计上 ,进行定量检测 ,分析了 3 6例慢性肾炎患者 ,1 8例尿毒症患者血浆中 1 2种游离氨基酸和 3 5例慢性肾炎患者尿液中 1 3种游离氨基酸 ,分别与 2 9例正常人血浆和3 0例正常人尿液中游离氨基酸进行了比较 ,以此探讨肾脏疾病体液氨基酸模式的同时提供氨基酸治疗肾脏疾病的科学依据。     

Evidence that DNA damage is associated to phenylalanine blood levels in leukocytes from phenylketonuric patients

Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism, biochemically characterized by the accumulation of Phe and its metabolites in blood and tissues of affected patients. Treatment for PKU consists of a protein restricted diet supplemented with a mixture containing essential amino acids (other than Phe) and micronutrients. In recent years several authors have studied the pathomechanisms of the disease and demonstrated the existence of lipid and protein oxidative damage in PKU patients. In this work we investigated the in vivo and in vitro effects of Phe on DNA damage determined by the alkaline comet assay using silver staining and visual scoring. We found a dose-dependent effect of Phe on DNA damage in leukocytes from normal individuals incubated with different concentrations of Phe. Additionally, by analyzing blood leukocytes from two groups of treated PKU patients based on their blood Phe levels, we verified that the DNA damage index was significantly higher in PKU patients with high Phe blood levels (DI = 68.2 ± 12.3), compared to well-treated patients and the control group (healthy individuals). Furthermore, well-treated PKU patients had greater DNA damage (DI = 44.9 ± 7.6) relatively to controls (DI = 12.7 ± 4.1). Our present in vitro and in vivo findings indicate that DNA damage occurs in peripheral blood from PKU patients and is associated to Phe blood levels.