Endo-Oligopeptidase A., a Putative Enkephalin-Generating Enzyme, in the Vertebrate Retina

Endo-oligopeptidase A, EC 3.4.22.19, converts small enkephalin-containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo-oligopeptidase. A found in pigeon retinae (30.3 +/- 7.3 mU/mg, mean +/- standard deviation) was four times higher than in rabbit retinae (7.0 +/- 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5,5'-dithiobis(2-nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo-oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double-labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo-oligopeptidase A-like immunoreactivity also contained [Leu5]-enkephalin-like immunoreactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo-oligopeptidase A in enkephalin generation in vivo.     

Inhibitors of a Rat Brain Enkephalin Aminopeptidase

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]- l -leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10⁻⁵ to 8.3 ± 10⁻⁸ m . The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.     

血管紧张素转换酶纯化与性质研究

为了深入了解猪肺血管紧张素转换酶 (angiotensin converting enzyme,ACE)的性质和功能 ,对猪肺 ACE的分离纯化以及部分酶学性质进行了研究 .猪肺组织匀浆经 1 .6～ 2 .6mol/L硫酸铵分级沉淀等步骤后 ,利用亲和胶进行亲和层析分离 .2 0 0 g猪肺组织中提纯出 0 .79mg ACE,比活力 38.8U/mg,SDS- PAGE电泳鉴定为一条带 ,分子量约 1 80 k D,等电点 (p I)为 p H4.5,糖含量约 2 3.6% ,氨基酸组成分析发现猪肺 ACE分子中含有 1 346个氨基酸 ,其中酸性氨基酸含量较高 ,碘乙酸的修饰结果表明猪肺 ACE中巯基基团未参与酶的催化反应 .酶反应动力学结果显示 ,ACE催化 Fa PGG底物反应时的最适 p H大约为 p H 7.6,反应活化能 Ea=4.37× 1 0 4 J/mol,酶活性部位附近的组氨酸和具有类似 α-氨基性质的氨基酸可能参与了 ACE催化反应 .有关猪肺 ACE的基本生化性质、氨基酸组成以及酶学性质的结果 ,为今后深入研究奠定了基础 .     

N-Acetylation of L-Aspartate in the Nervous System: Differential Distribution of a Specific Enzyme

L-Aspartate N-acetyltransferase, a nervous system enzyme that mediates the synthesis of N-acetyl-L-aspartic acid, has been characterized. In the presence of acetyl-CoA, L-aspartate was acetylated 10-fold more efficiently than L-glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10-fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10-15 pmol/min/mg tissue) and retina the lowest detectable activity (1-1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane-associated and was solubilized by treatment with Triton X-100.     

A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.     

Characterization of Neuronal and Endothelial Forms of Angiotensin Converting Enzyme in Pig Brain

The molecular forms of angiotensin converting enzyme (ACE; EC 3.4.15.1) in preparations of pig brain cortical microvessels and striatal synaptosomal membranes have been identified by immunoelectrophoretic blot analysis. The cortical microvessels contained only the endothelial form of the enzyme, Mr 180,000, which comigrated with pig kidney ACE on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, the synaptosomal membranes contained only a smaller form of ACE, Mr 170,000, which represents the neuronal form of the enzyme. No significant differences in inhibitor sensitivity or substrate specificity were detected between the two forms of ACE. In particular, neurokinin A was resistant to hydrolysis by either microvessel or synaptosomal membrane ACE, and the pattern of hydrolysis of substance P by the two preparations was identical.     

蛋氨酸脑啡肽质量标准研究

建立了蛋氨酸脑啡肽的质量标准。采用液相色谱-质谱联用仪检测蛋氨酸脑啡肽分子量和鉴别蛋氨酸脑啡肽的各氨基酸组成,反相高效液相色谱测定蛋氨酸脑啡肽含量。蛋氨酸脑啡肽分子量为573.7,氨基酸组成为Tyr-Gly-Gly-Phe-Met。蛋氨酸脑啡肽质量浓度在7~280 mg/L(r=0.999 8)内线性关系良好,平均回收率为98.54%,RSD为0.89%。所建立的方法科学,可靠,重复性好。可准确地对蛋氨酸脑啡肽进行定性定量检测。     

Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Escherichia coli d-Malate Dehydrogenase,a Generalist Enzyme Active in the Leucine Biosynthesis Pathway

The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of d-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on d-malate as a carbon source, the d-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB⁻ strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (l(+)-tartrate, d-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.     

Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme

Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34–72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR⁷²↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.     

Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties

The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with l-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial l-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low K_m property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of l-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes.     

4-Methylumbelliferyl-beta-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-beta-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity beta-N-acetylglucosaminidase-like enzyme (K(m), >100 mumol liter) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, beta-N-acetylglucosaminidase-like enzyme (K(m), <0.5 mumol liter). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r = 0.96) and Cyclidium sp. (r = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of beta-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Enzyme immobilization: an update

Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades. The most recent papers, patents, and reviews on immobilization strategies and application are reviewed.     

The Human Enzyme That Converts Dietary Provitamin A Carotenoids to Vitamin A Is a Dioxygenase

β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O₂ and H₂O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either ¹⁶O₂-H₂¹⁸O or ¹⁸O₂-H₂¹⁶O medium for 15 min at 37 °C, and the relative amounts of ¹⁸O-retinal and ¹⁶O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O₂ gas used. Together with the data from ¹⁸O-retinal-H₂¹⁶O and ¹⁶O-retinal-H₂¹⁸O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O₂ into retinal. Thus, BCO1 is a dioxygenase.     

A high-performance liquid chromatography assay for Dyrk1a,a Down syndrome-associated kinase

Down syndrome is the most common aneuploidy. It is caused by the presence of an extra copy of chromosome 21. Several studies indicate that aberrant expression of the kinase Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is implicated in Down syndrome, in particular in the onset of mental retardation. Moreover, elevated Dyrk1a activity may also be a risk factor for other neurodegenerative disorders such as Alzheimer’s disease. Over the past years, Dyrk1a has appeared as a potential drug target. Availability of sensitive and quantitative enzyme assays is of prime importance to understand the role of Dyrk1a and to develop specific inhibitors. Here, we describe a new method to measure Dyrk1a activity based on the separation and quantification of specific fluorescent peptides (substrate and phosphorylated product) by high-performance liquid chromatography (HPLC). Kinetic and mechanistic analyses using well-known inhibitors of Dyrk1a confirmed the reliability of this approach. In addition, this assay was further validated using brain extracts of mice models expressing different copies of the Dyrk1a gene. Our results indicate that this novel Dyrk1a assay is simple, sensitive, and specific. It avoids the use of radioactivity-based approaches that, until now, have been widely employed to measure Dyrk1a activity.     

1例雄性朱鹮脑内脑啡肽的免疫组织化学分布

用免疫组织化学方法研究脑啡肽(ENK)在极危物种朱(Nipponia nippon)脑内的分布,结合计算机图像分析仪检测免疫阳性细胞和末梢的灰度值。ENK阳性细胞、纤维和终末分布如下:发声核团有原纹状体中间区腹部、丘脑背内侧核外侧部、中脑丘间核、中脑背内侧核、延髓舌下神经核。听觉中枢有丘脑卵圆核壳区、中脑背外侧核壳区、脑桥外侧丘系腹核、上橄榄核、耳蜗核等。内分泌核团有视前区前核、旧纹状体增加部、下丘脑外侧核、下丘脑腹内侧核等。结果表明,朱脑内ENK可能对发声、听觉和下丘脑内分泌的生理活动有一定的调制作用。     

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.     

Ketomethylureas. A New Class of Angiotensin Converting Enzyme Inhibitors

Abstract

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term “ketomethylureas”, is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE wtih I₅₀ values in the nM range.     

Evolutionary Aspects of Enzyme Dynamics

The role of evolutionary pressure on the chemical step catalyzed by enzymes is somewhat enigmatic, in part because chemistry is not rate-limiting for many optimized systems. Herein, we present studies that examine various aspects of the evolutionary relationship between protein dynamics and the chemical step in two paradigmatic enzyme families, dihydrofolate reductases and alcohol dehydrogenases. Molecular details of both convergent and divergent evolution are beginning to emerge. The findings suggest that protein dynamics across an entire enzyme can play a role in adaptation to differing physiological conditions. The growing tool kit of kinetics, kinetic isotope effects, molecular biology, biophysics, and bioinformatics provides means to link evolutionary changes in structure-dynamics function to the vibrational and conformational states of each protein.