Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.     

Effect of wine and vinegar processing of Rhizoma Corydalis on the tissue distribution of tetrahydropalmatine, protopine and dehydrocorydaline in rats

Vinegar and wine processing of medicinal plants are two traditional pharmaceutical techniques which have been used for thousands of years in China. Tetrahydropalmatine (THP), dehydrocorydaline (DHC) and protopine are three major bioactive molecules in Rhizoma Corydalis. In this study, a simple and reliable HPLC method was developed for simultaneous analysis of THP, DHC and protopine in rat tissues after gastric gavage administration of Rhizoma Corydalis. The validated HPLC method was successfully applied to investigate the effect of wine and vinegar processing on the compounds' distribution in rat tissues. Our results showed that processing mainly affect the T(max) and mean residence time (MRT) of the molecules without changing their C(max) and AUC(0-24)( )(h) Vinegar processing significantly increased the T(max) of DHC in heart, kidney, cerebrum, cerebrellum, brain stem and striatum and prolonged the T(max) of protopine in brain. No significant changes were observed on the T(max) of THP in rat tissues after vinegar processing. Wine processing reduced the T(max) of protopine and DHC in liver and spleen and T(max) of protopine in lung, but increased the T(max) of THP in all the rat tissues examined. To our knowledge, this is the first report on the effects of processing on the tissue distribution of the bioactive molecules from Rhizoma Corydalis.     

Dual‐enzyme,co‐immobilized capillary microreactor combined with substrate recycling for high‐sensitive glutamate determination based on CE

A new method for high‐sensitive determination of glutamate was developed and evaluated based on CE by using dual‐enzyme co‐immobilized capillary microreactor combined with substrate recycling. The capillary microreactor was prepared by covalently co‐immobilizing glutamate dehydrogenase (GDH) and glutamic pyruvic transaminase (GPT) on the inner surface of a capillary and was characterized by SEM, ultraviolet‐visible spectroscopy, and fluorescence spectroscopy. The GDH‐GPT co‐immobilized capillary microreactor showed great stability and reproducibility. The apparent K_m for glutamate with GDH‐GPT coupled reaction was determined to be 0.61±0.06 mM but 2.56±0.24 mM when only GDH was immobilized. Glutamate determination was based on on‐column monitoring UV absorption at 340 nm of the reaction product reduced nicotinamide adenine dinucleotide, of which peak area was directly related to the glutamate concentration. The response of the present co‐immobilized GDH‐GPT assay for glutamate is greatly enhanced over single enzyme system, and a 15.7‐fold improvement in sensitivity was obtained. The detection limit of the proposed method is 0.15 μM glutamate (S/N=3). Selectivity for glutamate is good over most of the 20 amino acids. Finally, this method was successfully applied to determine the glutamate content in rat plasma and serum samples.     

Purification of human glutamate dehydrogenase (GDH) and an adsorptive voltammetric investigation of the interaction of GDH with rabbit anti-human GDH antibody

A procedure for the isolation of glutamate dehydrogenase (GDH) from human liver, which involves the use of ion-exchange chromatography on diethylaminoethyl cellulose and affinity chromatography on guanosine triphosphate conjugated to Sepharose 4B, is described. The adsorptive voltammetric behaviour of human GDH, bovine GDH and rabbit anti-human GDH antibody was optimised with respect to accumulation potential, accumulation time and scan rate. The lower limits of detection were 0.2 and 1.2 mg l-1 for human and bovine GDH, respectively, and the lower limit of detection for rabbit anti-GDH antibody was 0.04 mg l-1. The interaction of human GDH with rabbit anti-human GDH antibody was also examined using this method.     

RNA synthetic activity of glutamate dehydrogenase

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.     

Electrochemical study of the effect of ADP and AMP on the kinetics of glutamate dehydrogenase

A chronoamperometric method based on the 'diffusion' layer concept of the convective system was used to assay the glutamate dehydrogenase (GLDH) activity. Once the reaction was initiated by adding the enzyme GLDH into a well-stirred nicotinamide adenine dinucleotide (NADH, coenzyme) solution, the steady-state oxidation limiting current of NADH would decrease linearly in a short time. The major advantage of this method is that it directly indicates the continuous in-situ change of the coenzyme concentration, thus, the real initial reaction rate of enzyme-catalyzed reaction, V0, can be determined. Using this method, the effect of adenosine-5'-monophosphate (AMP) and adenosine-5'-diphosphate (ADP) on the GLDH activity has been monitored. The results showed that ADP and AMP could increase the activity of GLDH. This activation mechanism was proposed by the voltammetric study.     

Role of rare-earth elements in enhancing bioelectrocatalysis for biosensing with NAD+-dependent glutamate dehydrogenase

Dehydrogenases (DHs) are widely explored bioelectrocatalysts in the development of enzymatic bioelectronics like biosensors and biofuel cells. However, the relatively low intrinsic reaction rates of DHs which mostly depend on diffusional coenzymes (e.g., NAD⁺) have limited their bioelectrocatalytic performance in applications such as biosensors with a high sensitivity. In this study, we find that rare-earth elements (REEs) can enhance the activity of NAD⁺-dependent glutamate dehydrogenase (GDH) toward highly sensitive electrochemical biosensing of glutamate in vivo. Electrochemical studies show that the sensitivity of the GDH-based glutamate biosensor is remarkably enhanced in the presence of REE cations (i.e., Yb³⁺, La³⁺ or Eu³⁺) in solution, of which Yb³⁺ yields the highest sensitivity increase (ca. 95%). With the potential effect of REE cations on NAD⁺ electrochemistry being ruled out, homogeneous kinetic assays by steady-state and stopped-flow spectroscopy reveal a two-fold enhancement in the intrinsic reaction rate of GDH by introducing Yb³⁺, mainly through accelerating the rate-determining NADH releasing step during the catalytic cycle. In-depth structural investigations using small angle X-ray scattering and infrared spectroscopy indicate that Yb³⁺ induces the backbone compaction of GDH and subtle β-sheet transitions in the active site, which may reduce the energetic barrier to NADH dissociation from the binding pocket as further suggested by molecular dynamics simulation. This study not only unmasks the mechanism of REE-promoted GDH kinetics but also paves a new way to highly sensitive biosensing of glutamate in vivo.

This study demonstrated that REEs serve as allosteric promoters for bioelectrocatalysis of glutamate dehydrogenase by triggering subtle reorientation of peptide segments, consequently expediting phase coupling along with the catalytic scheme.     

Photomodulation of glutamate dehydrogenase properties by red light

To gain some insight into the mechanism by which red light-biosystem interaction occurs, an investigation was made of certain features of purified glutamate dehydrogenase from beef liver (E.C. 1.4.1.3.) irradiated with either an He---Ne laser (632.8 nm) or a red light-emitting diode (650±20 nm). In both cases the energy dose was 0.24 J cm⁻². Significant changes in the glutamate dehydrogenase extinction coefficient measured at 275 nm, the capability of the enzyme to bind the reduced form of nicotinamide adenine dinucleotide (NADH), certain kinetic parameters, the pH and temperature dependence and the sensitivity to guanosine 5 triphosphate (GTP) and adenosine diphosphate (ADP) were found, probably due to the interaction of lightwith a protein domain containing a metal ion or ions. He---Ne laser and diode irradiation were found to differ with regard to their interaction with glutamate dehydrogenase. Interestingly, different effects were also found when an He---Ne laser and a non-coherent Xe---Hg lamp were used to irradiate glutamate dehydrogenase under the same experimental conditions. This confirms that non-coherent light at various power levels affects the isolated glutamate dehydrogenase.     

Use of 1H-NMR metabolomics to precise the function of the third glutamate dehydrogenase gene in Arabidopsis thaliana

It is now well established that the GS/GOGAT cycle is the major route for ammonium assimilation in higher plants. However, it has often been argued that other enzymes, such as glutamate dehydrogenase, have the capacity to assimilate ammonium, leading to the hypothesis that alternative ammonium assimilatory pathways could operate under particular physiological conditions. The GDH enzyme is encoded by two distinct genes, GDH1 and GDH2. A third gene, GDH3, potentially encoding GDH has recently been identified by in silico studies performed on Arabidopsis thaliana. In order to precise its function, the metabolic profile of gdh3 knock out mutants were compared to wild type plants using the ¹H-NMR technique. ¹H-NMR spectra coupled with principal component analysis and partial least square-discriminant analysis were applied to identify changes of the metabolic profiles. These experiments were performed on roots, leaves and stems. In the gdh3 mutant, metabolic variations were observed for carboxylic acids, amino acids and carbohydrates content.     

Chingazumianine,a Novel Dichlorinated Alkaloid from Corydalis koidzumiana

A dichlorinated alkaloid with a novel skeleton, chingazumianine ( 1 ), was isolated from the herb Corydalis koidzumiana, and its structure elucidated by spectroscopic data and X‐ray crystallographic analysis. In citrate‐treated human platelet‐rich plasma (PRP), known compounds, i.e., protopine ( 2 ), (±)‐tetrahydropalmatine ( 3 ), and palmatine ( 4 ), isolated from this plant, showed significant inhibition of secondary aggregation induced by adrenaline in a concentration‐dependent manner, suggesting that the antiplatelet effects of these compounds is mainly due to an inhibitory effect on thromboxane formation.     

Phytochemical and biological activity studies of the Bhutanese medicinal plant Corydalis crispa

The chemical constituents and biological activities of Corydalis crispa (Fumariaceae) were investigated for the first time. The phytochemical study resulted in the isolation of nine known isoquinoline alkaloids: protopine (1), 13-oxoprotopine (2), 13-oxocryptopine (3), stylopine (4), coreximine (5), rheagenine (6), ochrobirine (7), sibiricine (8) and bicuculline (9), with complete NMR data for 2 and 3 provided here for the first time. Crude extracts exhibitedsignificant anti-inflammatory (p < 0.01) activity against TNF-alpha production in LPS activated THP-1 cells. The acetylcholinesterase inhibitory activity of compounds 2, 4 and 7 and the antiplasmodial activity of compound 5 against P. falciparum strains TM4/8.2 and K1CB1 (multidrug resistant strain) are reported here for the first time. Stylopine (4) did not show antimalarial activity against the K1CB1 strain in contrast to a previous report. This study generated a scientific basis for the use of this plant in Bhutanese traditional medicine, either individually or in combination with other medicinal ingredients to treat a broad range of disorders. This study also identified compound 5 as potential new antimalarial lead compound.     

Identification and Quantification,Metabolism and Pharmacokinetics,Pharmacological Activities,and Botanical Preparations of Protopine: A Review

Through pharmacological activity research, an increasing number of natural products and their derivatives are being recognized for their therapeutic value. In recent years, studies have been conducted on Corydalis yanhusuo W.T. Wang, a valuable medicinal herb listed in the Chinese Pharmacopoeia. Protopine, one of its components, has also become a research hotspot. To illustrate the identification, metabolism, and broad pharmacological activity of protopine and the botanical preparations containing it for further scientific studies and clinical applications, an in-depth and detailed review of protopine is required. We collected data on the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine from 1986 to 2021 from the PubMed database using “protopine” as a keyword. It has been shown that protopine as an active ingredient of many botanical preparations can be rapidly screened and quantified by a large number of methods (such as the LC-ESI-MS/MS and the TLC/GC-MS), and the possible metabolic pathways of protopine in vivo have been proposed. In addition, protopine possesses a wide range of pharmacological activities such as anti-inflammatory, anti-platelet aggregation, anti-cancer, analgesic, vasodilatory, anticholinesterase, anti-addictive, anticonvulsant, antipathogenic, antioxidant, hepatoprotective, neuroprotective, and cytotoxic and anti-proliferative activities. In this paper, the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine are reviewed in detail to lay a foundation for further scientific research and clinical applications of protopine.     

Nylon tube-immobilized creatinine iminohydrolase and glutamate dehydrogenase in serum and urine creatinine analysis

Immobilized enzyme nylon-tube reactors incorporating creatinine iminohyrolase (CI) and glutamate dehydrogenase (GDH) were used to assay creatinine in serum and urine. Optimum substrate concentrations for the assay were determined. The reactors were incorporated into a continuous flow system for creatinine analysis. The method was evaluated with respect to linearity, sample interaction, precision, accuracy, and analytical recovery. Comparison studies were carried out with a standard Jaffé method and the effect of interfering substances was investigated. From the results obtained, it was concluded that the assay was suitable as a simple, reliable, and specific method for serum and urine creatinine determinations.     

High-performance liquid chromatography-time-of-flight mass spectrometry with adjustment of fragmentor voltages for rapid identification of alkaloids in rat plasma after oral administration of rhizoma Corydalis extracts

A tandem high-performance liquid chromatography and time-of-flight mass spectrometry method has been developed for rapid separation and identification of alkaloids in rat plasma after oral administration of rhizoma Corydalis extracts. Compounds were separated on a Zorbax XDB-C(18) column and the linear gradient elution was used with the mobile phase consisting of ammonium acetate-0.01% acetic acid and acetonitrile. Mass spectra were acquired by electrospray ionization in the positive ion mode. Fifteen components were detected in vitro, among which 12 components were rapidly identified based on the accurate-mass capability of time-of-flight analyzer. Eleven prototype components were detected in dosed rat plasma (in vivo). Three isomers, which had the same [M+H](+) ion at m/z 356.1856, were distinguished as glaucine, tetrahydropalmatine, and corybulbine by dynamic adjustment of fragmentor voltages. This study developed a rapid and sensitive method that was successfully utilized for screening the active ingredients of rhizoma Corydalis in rat plasma, which provided helpful information for further pharmacokinetic research of active components in this medicine.     

New insights from X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example

A three-step process for immobilization of glutamate dehydrogenase (GDH) on the surface of silicon dioxide has been studied by X-ray photoelectron spectroscopy (XPS). The enzyme layer was deposited on the silicon dioxide surface after first exposing the surface to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surface with glutaraldehyde (GA). Fine XPS analysis, performed after each step of the chemical procedure, revealed unknown details of the step-by-step construction of the enzyme layer under different experimental conditions.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography

Positively charged reversed‐phase liquid chromatography was employed for the efficient preparative separation of isoquinoline alkaloids from Corydalis impatiens. Ten commercially available columns were compared for isoquinoline alkaloids analysis. While tailing, overloading, lower resolution, and buffer salts limited the application in purification of isoquinoline compounds of many of these columns, one positively charged reversed‐phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading isoquinoline compounds on a larger, preparative scale. The general separation process is as follows. First, isoquinoline alkaloids are enriched with Corydalis impatiens extract via a middle chromatogram isolated gel column. After column selection, separation is performed on an XCharge C18 analytical column, from which two evident chromatographic peaks are readily obtained. Finally, two isoquinoline alkaloids (protopine and corydamine) are selectively purified on the XCharge C18 preparative column. These results demonstrate that a middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography is effective for the preparative separation of isoquinoline alkaloids from Corydalis impatiens.     

Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Corydalis decumbens (Thunb.) Pers. using a multilayer coil planet centrifuge (CPC). The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)-acetonitrile-water (2:2:3, v/v) where triethylamine (5-10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5-10 mM) to the aqueous mobile phase as an eluter. From 3.1 g of the crude extract, 495 mg protopine, 626 mg tetrahydropalmatine and 423 mg bicuculline were obtained each with a purity of over 93% as determined by high performance liquid chromatography. The structures of the isolated compounds were identified by electron ionization mass spectrometry (EI-MS), high-performance liquid chromatography (HPLC)-electrospray ionisation-mass spectrometry (ESI-MS) and 1H NMR.     

Screening of antinociceptive components in Corydalis yanhusuo W.T. Wang by comprehensive two-dimensional liquid chromatography/tandem mass spectrometry

Formalin-induced pain models were used in rats to evaluate the antinociceptive effect of the total alkaloids of Corydalis yanhusuo (TAC). The results indicated that formalin-evoked spontaneous nociceptive responses (licking behavior) could be inhibited significantly by giving (intragingival) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed. The absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (seven compounds identified) were absorbed into the plasma with appropriate concentrations and about 20 compounds (four compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine, and corydaline) which were reported to possess profound antinociceptive effects exhibited high concentrations in the striatum. This may result from their synergistic effects in regulating the formalin-induced nociception. The results indicated that the comprehensive two-dimensional biochromatography method developed is capable of screening the bioactive components in Corydalis yanhusuo and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.     

Conventional sample enrichment strategies combined with high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance analysis allows analyte identification from a single minuscule Corydalis solida plant tuber

Identification of putative biomarker molecules within the genus Corydalis (Papaveraceae) was pursued by combining conventional off-line sample enrichment with high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) based structure elucidation. Off-line reversed phase solid phase extraction (SPE) was used to enrich the desired analytes from a methanolic extract (93 mg dry weight) of a miniscule single tuber (233 mg dry weight) of C. solida. An aliquot of the SPE fraction (2.1 mg) was subjected to separation in the HPLC-SPE-NMR hyphenation. Chromatographic peaks bearing the metabolites under investigation were trapped in the SPE device in a single experiment and transferred to a 600 MHz NMR spectrometer equipped with a 30 microl cryofit insert fed into a 3 mm cryoprobe. Recorded homo- and heteronuclear 1D and 2D NMR data allowed the identification of the three analytes under investigation as protopine, allocryptopine, and N-methyl-laudanidinium acetate. The latter is a rare alkaloid, which has been isolated only once before.     

Pharmacokinetics and tissue distribution of five bioactive components in the Corydalis yanhusuo total alkaloids transdermal patch following Shenque acupoint application in rats assessed by ultra-performance liquid chromatography–tandem mass spectrometry

The purpose of this study was to evaluate the pharmacokinetics and tissue distribution of the Corydalis yanhusuo total alkaloids transdermal patch (CTTP) following Shenque acupoint application in rats. The concentrations of corydaline, tetrahydropalmatine, tetrahydrocolumbamine, protopine, and dehydrocorydaline in rat plasma and various tissues were simultaneously detected by ultra-performance liquid chromatography–tandem mass spectrometry after Shenque acupoint administration of CTTP. Plasma, heart, liver, spleen, lung, and kidney tissue samples were collected at specific times and separated by gradient elution on an ACQUITY UPLC HSS T3 column (1.8 μm, 100 mm × 2.1 mm) with a mobile phase of 0.01% formic acid aqueous solution and acetonitrile–0.01% formic acid. The methodological results showed that the selectivity, linear range, accuracy, precision, stability, matrix effect, and extraction recovery of the established method met the requirements of biological sample analysis. The results indicated that CTTP following Shenque acupoint administration rapidly delivered adequate drug into rat blood and maintained an effective plasma level for a significantly longer time than non-acupoint administration. Furthermore, CTTP effectively reached the liver through Shenque acupoint administration and showed tissue selectivity. The data obtained could provide a prospect for the treatment of chronic pain with CTTP following Shenque acupoint application.