BIOSYNTHESIS OF THE COUMARINS: III. THE ROLE OF GLYCOSIDES IN THE FORMATION OF COUMARIN BY HIEROCHLOE ODORATA

1962 ◽  
Vol 40 (5) ◽  
pp. 607-618 ◽  
Author(s):  
Stewart A. Brown
Keyword(s):  
Specific Activity ◽  
Indirect Evidence ◽  
Coumaric Acid ◽  
Major Pathway ◽  
Metabolic Intermediates ◽  
Simple Extraction ◽  
Hierochloe Odorata ◽  
Hydrolysis Of

Coumarinic acid β-glucoside has been identified in Hierochloe odorata. Carbon dioxide-C14was administered to this species, and the plants were allowed to metabolize for periods up to 31 days. "Free" coumarin was recovered by simple extraction, and additional coumarin and o-coumaric acid were recovered after hydrolysis of the corresponding glucosides with emulsin. The total C14in o-coumaric acid reached a maximum in about 4 days and then declined, but the peak in the total C14of coumarin was not reached until 8 to 17 days after activation. o-Coumaryl glucoside and coumarin are thus both metabolic intermediates rather than end products, and the glucoside is formed first. This supports the orthohydroxylation theory of coumarin biosynthesis. The specific activity of coumarin liberated by emulsin hydrolysis from the glucoside was consistently lower than that of "free" coumarin. This is irreconcilable with the proposed formation of free coumarin from coumarinic acid glucoside as a major pathway in vivo unless separate pools of the glucoside exist in the plant. Indirect evidence is presented that free coumarin can exist in Hierochloe.

BIOSYNTHESIS OF THE COUMARINS: III. THE ROLE OF GLYCOSIDES IN THE FORMATION OF COUMARIN BY HIEROCHLOE ODORATA

1962 ◽  
Vol 40 (1) ◽  
pp. 607-618 ◽  
Author(s):  
Stewart A. Brown
Keyword(s):  
Specific Activity ◽  
Indirect Evidence ◽  
Coumaric Acid ◽  
Major Pathway ◽  
Metabolic Intermediates ◽  
Simple Extraction ◽  
Hierochloe Odorata ◽  
Hydrolysis Of

Coumarinic acid β-glucoside has been identified in Hierochloe odorata. Carbon dioxide-C14was administered to this species, and the plants were allowed to metabolize for periods up to 31 days. "Free" coumarin was recovered by simple extraction, and additional coumarin and o-coumaric acid were recovered after hydrolysis of the corresponding glucosides with emulsin. The total C14in o-coumaric acid reached a maximum in about 4 days and then declined, but the peak in the total C14of coumarin was not reached until 8 to 17 days after activation. o-Coumaryl glucoside and coumarin are thus both metabolic intermediates rather than end products, and the glucoside is formed first. This supports the orthohydroxylation theory of coumarin biosynthesis. The specific activity of coumarin liberated by emulsin hydrolysis from the glucoside was consistently lower than that of "free" coumarin. This is irreconcilable with the proposed formation of free coumarin from coumarinic acid glucoside as a major pathway in vivo unless separate pools of the glucoside exist in the plant. Indirect evidence is presented that free coumarin can exist in Hierochloe.

scholarly journals A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

2010 ◽  
Vol 192 (18) ◽  
pp. 4776-4785 ◽  
Author(s):  
Rabeb Dhouib ◽  
Françoise Laval ◽  
Frédéric Carrière ◽  
Mamadou Daffé ◽  
Stéphane Canaan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Cell Interaction ◽  
Monoacylglycerol Lipase ◽  
Homologous Proteins ◽  
Dependent Activity ◽  
Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

Influence of C-ANF receptor and neutral endopeptidase on pharmacokinetics of ANF in rats

1991 ◽  
Vol 260 (1) ◽  
pp. R208-R216 ◽  
Author(s):  
P. J. Chiu ◽  
G. Tetzloff ◽  
M. T. Romano ◽  
C. J. Foster ◽  
E. J. Sybertz
Keyword(s):  
High Performance ◽  
Fold Increase ◽  
Neutral Endopeptidase ◽  
Volume Of Distribution ◽  
Fourfold Increase ◽  
Control Value ◽  
Enzymatic Pathways ◽  
Hydrolysis Of

The role of C-atrial natriuretic factor (ANF) receptors and neutral endopeptidase (NEP) in the pharmacokinetics and hydrolysis of 125I-labeled ANF was evaluated in rats by using C-ANF and SCH 39370 to block the nonenzymatic and enzymatic pathways, respectively. After a bolus injection of 125I-ANF, the resulting area under the plasma concentration curve (AUC) with C-ANF treatment was seven times the control value with regard to trichloroacetic acid-precipitable (TCA-ppt) radioactivity (intact ANF). SCH 39370 tended to increase AUC, but the changes were not significant. Nevertheless, SCH 39370 suppressed the appearance of TCA-soluble radioactivity (hydrolytic products), indicating that in vivo inhibition of ANF degradation had occurred. SCH 39370 plus C-ANF produced a 15-fold increase in AUC for TCA-ppt radioactivity and a reduction in plasma TCA-soluble radioactivity. High-performance liquid chromatography (HPLC) analysis confirmed that combination treatment increased intact ANF and reduced hydrolytic products in the plasma. SCH 39370 reduced clearance (C) without altering volume of distribution in steady state (Vss) and half-life (t1/2). C-ANF decreased both C and Vss leading to a fourfold increase in t1/2, which was further prolonged by SCH 39370 (7.5 times control). Bilateral nephrectomy caused a proportionally similar decrease in Vss and C without changing t1/2, suggesting significant extrarenal metabolism of ANF. SCH 39370 systemically inhibits ANF hydrolysis; the resulting increase in ANF, however, is masked by the great capacity of ANF clearance receptors but can be revealed with excess C-ANF, suggesting that the plasma ANF concentrations are determined by the interplay of the C-ANF receptor and NEP systems.

Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

1996 ◽  
Vol 271 (4) ◽  
pp. H1340-H1347 ◽  
Author(s):  
A. Decarie ◽  
P. Raymond ◽  
N. Gervais ◽  
R. Couture ◽  
A. Adam
Keyword(s):  
Metabolic Pathways ◽  
Neutral Endopeptidase ◽  
Thrombin Inhibitor ◽  
Rat Serum ◽  
Highly Sensitive ◽  
Significant Difference ◽  
Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

scholarly journals Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

2005 ◽  
Vol 4 (6) ◽  
pp. 1057-1065 ◽  
Author(s):  
M. Wilhelm ◽  
F.-X. Wilhelm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Specific Activity ◽  
Rnase H ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Acidic Domain ◽  
Basic Region

ABSTRACT Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

scholarly journals Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State

2008 ◽  
Vol 190 (21) ◽  
pp. 7302-7307 ◽  
Author(s):  
Sanchaita Das ◽  
Elizabeth Stivison ◽  
Ewa Folta-Stogniew ◽  
Donald Oliver
Keyword(s):  
Cell Growth ◽  
Functional State ◽  
Protein Translocation ◽  
Specific Activity ◽  
Cell Fractionation ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Carboxyl Terminal

ABSTRACT The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecAΔ8 lacking residues 2 to 8, SecAΔ11 lacking residues 2 to 11, and SecAΔ11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecAΔ11 or SecAΔ11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecAΔ11 and SecAΔ11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecAΔ11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecAΔ11/N95 mutant than of the SecAΔ11 mutant. Biochemical studies showed that SecAΔ11 and SecAΔ11/N95 had identical dimerization defects, while SecAΔ8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecAΔ11 and SecAΔ11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

Elaboration of the 6,7,8 Oxygenation Pattern in Simple Coumarins: Biosynthesis of Puberulin in Agathosma puberula Fourc

1984 ◽  
Vol 39 (1-2) ◽  
pp. 31-37 ◽  
Author(s):  
Stewart A. Brown ◽  
D. E. A. Rivett ◽  
H. Joan Thompson
Keyword(s):  
Ferulic Acid ◽  
Hydroxycinnamic Acid ◽  
Coumaric Acid ◽  
Major Pathway

Abstract Umbelliferone and scopoletin are well utilized as precursors of puberulin, a 7-O-prenyl ether of isofraxidin elaborated by Agathosm a puberula Fourc. (Rutaceae). As ferulic, sinapic, and caffeic acids were all more poorly utilized than 4′-hydroxycinnamic (p-coumaric) acid, the partial biosynthetic route: 4′-hydroxycinnamic acid →→ um belliferone → aesculetin → scopoletin →→ puberulin is suggested as the major pathway. Prenylether formation apparently occurs at or beyond the scopoletin stage. The implication that ferulic acid does not participate in the formation of scopoletin from 4′-hydroxycinnamic acid is at variance with the known role of ferulic acid as a scopoletin precursor in tobacco.

scholarly journals Monoamine Oxidase Activity in the Cerebral Vasculature: Comparison between Fresh Microvessels from Different Structures and Cell Cultures Derived from Microvessels

1984 ◽  
Vol 4 (3) ◽  
pp. 415-424 ◽  
Author(s):  
R. Sercombe ◽  
F. Lasbennes ◽  
L. Drouet ◽  
A. M. Dosne ◽  
J. Seylaz
Keyword(s):  
Monoamine Oxidase ◽  
Caudate Nucleus ◽  
Cell Cultures ◽  
Regional Differences ◽  
Specific Activity ◽  
Synthetase Activity ◽  
Mao Activity ◽  
Main Factor

Monoamine oxidase (MAO) activity was studied in various preparations of porcine brain microvessels to explore further the role of this enzyme in the blood–brain barrier to catecholamines. No difference was noted ( Vm and Km) between microvessels isolated from three structures (caudate nucleus, thalamus, and cerebral cortex) in which the responses to circulating catecholamines in vivo are markedly different. Large and small microvessels from the caudate nucleus and the thalamus presented the same specific activity. Cell cultures obtained from small microvessels were rich in endothelial cells as identified by the presence of Factor VIII–related antigen. These preparations displayed an MAO activity about ninefold less than freshly isolated microvessels, although their prostaglandin synthetase activity appeared normal. These results suggest that MAO activity is not the main factor determining the regional differences in the cerebrovascular reactions to catecholamines, that MAO is not specifically localized in the endothelium but must be also present in the smooth muscle, and that the MAO activity is greatly decreased during cell culture.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

scholarly journals 14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

1998 ◽  
Vol 18 (7) ◽  
pp. 3947-3955 ◽  
Author(s):  
Sandrine Roy ◽  
Robert A. McPherson ◽  
Ann Apolloni ◽  
Jun Yan ◽  
Annette Lane ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Specific Activity ◽  
Membrane Recruitment ◽  
Oncogenic Ras ◽  
Epidermal Growth

ABSTRACT 14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.

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