Comprehensive evaluation of the composition of Mingshan Laochuancha green tea and demonstration of hypolipidemic activity in a zebrafish obesity model

Laochuancha is an ancient tea plant originating from the Mingshan district of Ya''an city, Sichuan province, China, which is used to produce tea products with excellent quality. Mingshan Laochuancha green tea (MLGT) is a type of green tea manufactured from Laochuancha tea leaves. Currently, not much is known regarding the chemical compositions of MLGT and its bioactivity. Herein, the present study explores, for the first time, the chemical compositions and hypolipidemic activity of MLGT. It was observed that K was the most abundant element of 26.58 mg g⁻¹, and contents of toxic As, Cd, Cr and Pb elements were all below concentration limits. Alcohols (55.65%) were the main volatiles, and numerous volatiles with chestnut-like aroma were detected. Total content of 21 amino acids was 28.61 mg g⁻¹, and amino acids with velvety-like and umami taste totally accounted for 65.39%. The high content of amino acids and low ratio of polyphenols to total amino acids were attributed to strong umami and mellow taste of MLGT. Moreover, catechins and alkaloids were abundant in MLGT, where EGCG (85.82 mg g⁻¹) and caffeine (33.78 mg g⁻¹) were at highest content. Analyses of chemical compositions revealed excellent quality of MLGT. Correspondingly, MLGT showed potent hypolipidemic activity, and water extract of MLGT at 200 μg mL⁻¹ significantly reduced lipid level to 43.06% of high-fat zebrafish. Results firstly revealed the quality characteristics of MLGT and provided further insights into bioactivity of Laochuancha.

MLGT was investigated for the first time, and results revealed excellent quality and potent hypolipidemic activity of MLGT.     

Cloning and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm, Bombyx mori

A serine protease of the silkworm, Bombyx mori, with an isoelectric point of pH 10–11 and a pH optimum for succinyl-Leu-Leu-Val-Tyr-MCA degrading activity of about 10, was found in a 0.33 m NaCI-eluted fraction obtained from cation-exchange chromatography of digestive juice. The activity of the enzyme was strongly inhibited by chymostatin and PMSF, indicating that the protease is a chymotrypsin-like serine protease. The N-terminal amino acid sequence of the protease was determined, and a full-length cDNA clone (0.92 kbp) which was isolated from a midgut cDNA library was sequenced. The cDNA encodes a pre-proenzyme of 284 amino acids with a pro-segment of 50 amino acids and mature protein of 234 amino acids. From its primary structure, the predicted molecular mass of the mature protein is 24.5 kDa. A sequence comparison of the Bombyx highly basic protease with other serine proteases revealed that this enzyme is a mammalian-type serine protease with a catalytic triad consisting of His⁴⁵, Asp⁹² and Ser¹⁸⁶. A large number of Arg residues are encoded by the cDNA which may be responsible for its stability and/or function in the alkaline condition, by remaining charged at high pH.     

The role of glucose,long-chain triglycerides and amino acids for promotion of amino acid balance across peripheral tissues in man

The role of amino acids, glucose and lipids in improving amino acid balance in peripheral tissues was evaluated. Primed constant infusion of L -[ring-²H₅]phenylalanine in combination with flux measurements of glucose, free fatty acids (FFA) and amino acids across arm and leg tissues were applied in male volunteers after an overnight fast with subsequent primed constant infusions of amino acids (0·2 g N kg^?1 body weight day^?1), long-chain triglycerides (0·98–1·079 g kg^?1 day^?1) and glucose (3·13–3·62 g kg^?1 day^?1). Amino acids and phenylalanine tracer infusion continued for 6 h; the lipid infusion was provided during 2–6 h from the start, and glucose infusion was provided between 4 and 6 h. Flux measurements were performed at steady state before the next infusion started. Arterial concentrations of infused substrates increased during provision, but remained constant thereafter. Plasma insulin increased when glucose was provided, whereas insulin-like growth factor (IGF) I was unchanged during all infusions. Blood flow was unchanged in arm tissue during all infusions, while leg blood flow increased during fat and glucose infusion. FFA and glucose balance were unchanged during amino acid infusion but improved during lipid and glucose infusions. Amino acid balance was negative across arm and leg tissues in the fasted state, but reached balance during amino acid infusion. This effect was equally dependent on protein synthesis and protein degradation without any contribution from lipids and glucose. 3-Methylhistidine release from tissues was not influenced by any substrate. Our results suggest that extracellular amino acid concentrations determine amino acid balance across peripheral tissues independently of non-protein calories, insulin and IGF-I.     

Towards a selective synthetic route for cobalt amino acid complexes and their application in ring opening polymerization of rac-lactide

Catalysts based on cobalt amino acids and 2,2 bipyridine (bipy) present an attractive and cost-effective alternative as ring opening polymerization catalysts, yet this system remains underexplored despite the advantageous coordination properties of amino acids and bipy as ligands combined with the variety of accessible oxidation states and coordination geometries of cobalt. Here, metal complexes of type [Co(aa)₂(bipy)] with amino acids (aa: glycine, leucine and threonine) as ligands are reported. The complexes were characterized spectroscopically (IR, UV-vis and ¹H, ¹³C NMR for diamagnetic species), and by MS spectrometry and elemental analysis. The data reveal that the 2,2 bipyridine acts as a neutral bidentate donor coordinating to the metal ion through two nitrogen atoms and the amino acid acts as a bidentate ligand coordinating through the carboxylate and amino group forming a stable five membered ring and a pseudo-octahedral geometry around the Co center. The activity of the complexes for the ring opening polymerization (ROP) of rac-lactide is presented. The complexes are effective initiators for the ROP of rac-lactide (K_obs = 9.05 × 10⁻⁴ s⁻¹) at 100 : 1 [rac-lactide] : [catalyst] 1 M overall concentration of lactide in toluene at 403 K.

Catalysts based on Co, amino acids, and 2,2-bipyridine present an attractive and economic alternative in ring opening polymerization, and possess advantageous ligand coordination properties combined with a variety of accessible oxidation states and coordination geometries.     

Metabolic,thermal and circulatory effects of intravenous infusion of amino acids in tetraplegic patients

Summary. Metabolic, circulatory and thermal effects of intravenously (i.v.) administered amino acids were studied in eight patients with complete cervical spinal cord injuries, and compared with the effects in eight healthy subjects. Using indirect calori-metry and catheter techniques, whole-body and splanchnic oxygen consumption, blood flow and blood temperatures were measured before and at timed intervals during 2.5 h of i.v. infusion of 600 kJ of a mixture of 19 amino acids. Pulmonary oxygen uptake increased from 209±11 to 267±13 ml min^-1 in the patients and from 268±5 to 320±8 ml min^-1 in the controls. The thermic effect of amino acids was 21±3% and 16±2% in patients and controls, respectively. In both groups the splanchnic tissues accounted for approximately half of the rise in whole-body oxygen consumption. Cardiac output rose by, on average, 0.5±0-l and 0.8±0.2 1 min^-1 in patients and controls, respectively, while the hepatic blood flow remained unchanged in both groups. Pulmonary arterial blood temperature increased by 0–647±0100°C in the patients and by 0.244±0.174°C in the controls (P<0.05). The whole-body specific heat was low in the patients, its calculated maximum value being approximately 20% below the normal level. During the amino acid infusion the arterial blood concentration of amino acids rose by approximately 170% and 112% of its basal levels in patients and controls, respectively, indicating a significantly reduced capacity for cellular uptake of amino acids in tetraplegic patients. It is concluded that, in tetraplegic patients, i.v. infused amino acids induce prompt thermogenesis of normal magnitude accompanied by supranormal temperatures and amino acid concentrations in the blood, and that low whole-body specific heat contributes to the well-known thermoregulatory instability in tetraplegia.     

Molecular Mode of Action of the Antifungal β-Amino Acid BAY 10-8888

BAY 10-8888 is a cyclic β-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H⁺-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a K_T (Michaelis constant of the transport reaction) of 0.95 mM and a V_max of 18.9 nmol × min⁻¹ × 10⁷ cells⁻¹. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.     

The effect of glutamine on protein balance and amino acid flux across arm and leg tissues in healthy volunteers

Background. Glutamine is important in nitrogen transportation and the physiological control of acid–base regulation. In addition, it has been assumed that glutamine regulates protein balance in skeletal muscles based on findings in both experimental and clinical studies. However, little information on glutamine and its effect on protein dynamics in normal individuals is available. Therefore, the aim of this study was to evaluate whether glutamine improves protein balance and uptake of various indispensable amino acids across peripheral tissue in healthy individuals. Material and methods. Standard primed constant infusions of L ‐[ring‐²H₅]phenylalanine and [ring 3,3‐²H₂]tyrosine (2 μmol kg^?1 h^?1) were performed after overnight fast in five healthy male volunteers before and during infusions of a standard and a glutamine/tyrosine enriched amino acid solution. Flux measurements of amino acids (AA) including 3‐methylhistidine, glucose, lactate and free fatty acids (FFA) were performed across arm and leg tissues. Results. Infusion of the standard AA solution (0·2 g N kg^?1 day^?1) increased the net uptake of individual amino acids, but provision of the enriched solution (0·4 g N kg^?1 day^?1) with increased amounts of glutamine and tyrosine seemed to compete unfavourably with the net uptake of other key amino acids as methionine and phenylalanine, which are indispensable in muscles for protein synthesis. Increased flux of amino acids across peripheral tissues did not influence on flux of glucose, free fatty acid and lactate. Conclusions. Glutamine provision did neither stimulate protein synthesis nor attenuate breakdown of either globular or myofibrillar proteins in skeletal muscles of healthy volunteers.     

Reprogramming natural proteins using unnatural amino acids

Unnatural amino acids have gained significant attention in protein engineering and drug discovery as they allow the evolution of proteins with enhanced stability and activity. The incorporation of unnatural amino acids into proteins offers a rational approach to engineer enzymes for designing efficient biocatalysts that exhibit versatile physicochemical properties and biological functions. This review highlights the biological and synthetic routes of unnatural amino acids to yield a modified protein with altered functionality and their incorporation methods. Unnatural amino acids offer a wide array of applications such as antibody-drug conjugates, probes for change in protein conformation and structure–activity relationships, peptide-based imaging, antimicrobial activities, etc. Besides their emerging applications in fundamental and applied science, systemic research is necessary to explore unnatural amino acids with novel side chains that can address the limitations of natural amino acids.

Incorporation of unnatural amino acids into protein offers wide array of applications in fundamental and applied science.     

Renal reserve filtration capacity in growth hormone deficient subjects

Abstract. In normal subjects, the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) acutely increase in response to infusion of amino acids and to low doses of dopamine. It is uncertain whether circulatory growth hormone (GH) is a permissive factor for these stimulatory effects. GFR and ERPF (constant infusion technique using ¹²⁵I-iothalamate and ¹³¹I-hippuran, respectively) were measured before and during the infusion of dopamine and amino acids in 8 GH deficient subjects. The clearance study was repeated during concomitant administration of octreotide to investigate whether this somatostatin analogue would modify the amino acid and dopamine-induced renal haemodynamic changes. Dopamine increased baseline GFR from 89± 3 (mean±SEM, n= 8) to 102 ± 4 ml min^-1 1.73 m^-2 and ERPF from 352± 19 to 476± 26 ml min^-1 1.73 m^-2, P<0.001 for both. During amino acid infusion GFR and ERPF increased to 108±3 and 415±23 ml min^-1 1.73 m^-2, respectively, P< 0.001 for both. Octreotide did not significantly decrease baseline and dopamine-stimu-lated renal haemodynamics but lowered the amino acid-stimulated GFR (98±4 ml min^-1 1.73 m^-2, P<0.05) and ERPF (381± 18 ml min^-1 1.73 m^-2, P<0.05). Basal plasma glucagon concentrations were not suppressed by octreotide, whereas the amino acid-induced increments in plasma glucagon were partially inhibited. It is concluded that GH is not a necessary factor for the stimulatory effects of amino acids and dopamine on renal haemodynamics. The renal reserve filtration capacity in GH deficiency was at least as large as previously documented in normal subjects. It is likely that there is a functional antagonism between the effects of amino acids and octreotide on renal haemodynamics in GH deficiency.     

The separate and combined effect of leucine and insulin on muscle free amino acids

Summary. The effect of insulin and leucine on amino acid and protein metabolism in muscle is not fully understood. To characterize their separate and combined effects on free amino acids in muscle and plasma, 11 volunteers received an infusion of either leucine (1 g h^-1, Group 1) or glucose (20 g h^-1, Group 2) for 2 h followed by a combination of the two infusions for an additional 2-h period. In muscle both the leucine infusion and the leucine plus glucose infusion increased the concentration of free leucine significantly, while the sum of the other branched chain amino acids (BCAA), of the aromatic amino acids and of the basic amino acids decreased. Glucose infusion alone decreased the sum of the essential amino acids, the BCAA and the aromatic amino acids. The combination of leucine and glucose augmented the decreases, while the concentrations of glutamate, glutamine and alanine were unaffected. In plasma the leucine infusion doubled the leucine concentration and decreased alanine, valine, methionine, tyrosine, phenylalanine and the sum of the aromatic amino acids. Glucose infusion decreased methionine, serine, isoleucine and the sum of the essential amino acids and of the BCAA. The combination of leucine infusion and hyperinsulinaemia augmented the decreases. The plasma concentrations of the keto acids of valine and isoleucine decreased by the leucine infusion while the concentrations of the keto acid of leucine and isoleucine decreased by glucose infusion. The combination of leucine and glucose had an additive effect. These effects are attributed to a specific effect of leucine on the other two BCAA and a depression of muscle proteolysis by both leucine and insulin, resulting from glucose infusion.     

Melting properties of amino acids and their solubility in water

The state-of-the-art unit operation for separation and purification of amino acids is still crystallization, which requires solubility data and melting properties of pure compounds. Since measuring solubility is time-consuming, prediction tools are desired. Further, melting properties are not yet available due to decomposition of amino acids upon slow heating. In this work, melting properties of twenty amino acids (except Met) were measured by Fast Scanning Calorimetry (FSC) with heating rates up to 20 000 K s⁻¹. PC-SAFT was used to predict interactions in amino acid + water systems. Additionally, solubility, pH, and PXRD was measured. By combining FSC and PC-SAFT, the solubility of 15 amino acids was successfully predicted in a wide temperature range in good agreement with the experimental data. Thus, this work provides melting properties of amino acids for the first time and highlights the usefulness of such data to predict material properties such as aqueous solubility of amino acids.

We report the melting properties of amino acids for the first time and highlight the usefulness of such data to predict material properties such as aqueous solubility of amino acids.     

Leucine Oxidation and Protein Turnover in Clofibrate-induced Muscle Protein Degradation in Rats

Treatment of hyperlipidemia with clofibrate may result in development of a muscular syndrome. Our previous investigation (1979. J. Clin. Invest.64: 405.) showed that chronic administration of clofibrate to rats causes myotonia and decreases glucose and fatty acid oxidation and total protein of skeletal muscle. In the present experiments we have investigated amino acid and protein metabolism in these rats. Clofibrate administration decreased the concentration of all three branched-chain amino acids without affecting those of others in muscle. Studies to examine the mechanism of decreases in muscle concentrations of branched-chain amino acids showed the following: (a) Plasma concentration of leucine was decreased, whereas there was no significant change in the concentration of isoleucine and valine. (b) Liver concentrations of all three branched-chain amino acids remained unaltered. (c) The uptake of cycloleucine (a nonmetabolizable analogue of leucine) by both muscle and liver was unaffected. (d) The percentage of a trace amount of injected [1-¹⁴C]leucine expired as ¹⁴CO₂ in 1 h was significantly increased. (e) The capacity of muscle homogenate for α-decarboxylation of leucine was enhanced, whereas that of liver was unaffected. (f) The activity of leucine transaminase was unaffected, whereas that of α-ketoisocaproate dehydrogenase was increased in muscle.     

A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity

The plasma membrane Na⁺/Ca²⁺ exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na⁺ ions for 1Ca²⁺ ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP_NCX1), represent an autoinhibitory region involved in the Na⁺-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP_NCX1 region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562–688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP_NCX1 domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.     

The effect of hypoxia on the metabolism of labeled glucose and acetate in the rat brain

Glucose consumption and utilization of amino acids, lipids and proteins was measured in the rat brain under normoxia and hypoxia (70%N₂: 93%N₂). After 2 h of hypoxia the brain glucose consumption increased by over 60% of control value. In spite of this increase, the radioactivity of amino acid fraction did not increase or parallel changes of glucose radioactivity in the blood. This strongly suggested that glucose flux into amino acids remained unchanged in hypoxia. Incorporation of °C from glucose into macromolecules was found to decrease. The above changes demonstrated that the metabolic steps which precede synthesis of amino and tricarboxylic acids were inhibited. In some experiments, the incorporation of ⁴C from [2-¹⁴C]-acetate into a macromolecular fraction was also measured. The amounts of radioactivity found in these fractions were unchanged under hypoxic conditions.Possible differences in the influence of hypoxia on macromolecular synthesis in different metabolic compartments of the brain are discussed.     

Placental membrane transport

Brush border (microvillous) plasma membranes (BBM) and the basal surfaces (BCM) from the syncytiotrophoblast of human term placenta were prepared by a method of sonication, dialysis and differential centrifugation in specific buffer systems. Such plasma membranes formed closed, osmotically active, right-side-out vesicles in which amino acid transport could be studied unidirectionally by a carefully designed membrane filtration assay under reduced pressure. In such vesicles,l-leucine (1 mM) was found to be transported in a time-dependent manner, peak accumulation being attained at 45 s in both BBM and BCM. The accumulation of leucine in the vesicles was dependent on an inward NaCl gradient, as replacing the Na⁺with K⁺, Li⁺and choline, or replacing the Cl^? with SO ₄ ^2? failed to influence the amino acid movement. Leucine transport in the vesicles also appeared to be dependent on the substrate concentration, indicating saturation at a higher concentration. The transport process showed a k_t (affinity constant) of 3.85 and 6.67 mM, while the values recorded for the J_max (maximum apparent initial velocity) were 270.27 and 384.62 nmol/mg protein^?1 per min in the BBM and BCM respectively. Leucine transport was inhibited by a number of amino acids, among which amino isobutyric acid (AIB) produced the maximum inhibition. The k_i (inhibition constant) for different amino acids has also been listed. Lysine showed the least k_i value, thus showing it to be the most inhibitory compound. These findings are discussed in relation to the mechanism and regulation of transplacental amino acid transfer.     

Metabolic abnormalities of amino acids in patients with alcoholic liver damage]

The metabolic abnormality of amino acids in patients with alcoholic liver damage is discussed. The abnormalities are variable according to the degree of hepatic damage, the amount of alcohol consumption, and the intake of amino acids. In general, the plasma concentration of branched-chain amino acids, aromatic amino acids and alpha-amino-n-butyric acid increases, whereas that of hydroxy amino acids, alanine and proline decreases, in alcoholics with liver damage. Serum gamma-glutamyltranspeptidase activity increases with the increase of alcohol consumption. This increase is accentuated by lowered carbohydrate intake at the time of alcohol ingestion. A nutritional survey among healthy male subjects revealed that the intake of cereals decreases with increasing amount of alcohol consumption, a finding which suggests that the intake of some amino acids may be altered by drinking habit.     

Imaging Tumour ATB0,+ Transport Activity by PET with the Cationic Amino Acid O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine

Purpose

The concentrative amino acid transporter ATB^0,+ (SLC6A14) is under evaluation as a target for anticancer therapy. An ATB^0,+-selective positron emission tomography (PET) probe could advance preclinical drug development. We characterised the cationic tyrosine analogue O-2((2-[¹⁸F]fluoroethyl)methyl-amino)ethyltyrosine ([¹⁸F]FEMAET) as a PET probe for ATB^0,+ activity.

Procedures

Cell uptake was studied in vitro. ATB^0,+ expression was quantified by real-time PCR. [¹⁸F]FEMAET accumulation in xenografts was investigated by small animal PET with mice.

Results

[¹⁸F]FEMAET accumulated in PC-3 and NCI-H69 cancer cells in vitro. As expected for ATB^0,+ transport, uptake was inhibited by LAT/ATB^0,+ inhibitors and dibasic amino acids, and [¹⁸F]FEMAET efflux was only moderately stimulated by extracellular amino acids. ATB^0,+ was expressed in PC-3 and NCI-H69 but not MDA-MB-231 xenografts. PET revealed accumulation in PC-3 and NCI-H69 xenografts and significant reduction by ATB^0,+ inhibition. Uptake was negligible in MDA-MB-231 xenografts.

Conclusion

ATB^0,+ activity can be imaged in vivo by PET with [¹⁸F]FEMAET.     

New marine-derived indolymethyl pyrazinoquinazoline alkaloids with promising antimicrobial profiles

Due to the emergence of multidrug-resistant pathogenic microorganisms, the search for novel antimicrobials is urgent. Inspired by marine alkaloids, a series of indolomethyl pyrazino [1,2-b]quinazoline-3,6-diones was prepared using a one-pot microwave-assisted multicomponent polycondensation of amino acids. The compounds were evaluated for their antimicrobial activity against a panel of nine bacterial strains and five fungal strains. Compounds 26 and 27 were the most effective against Staphylococcus aureus ATCC 29213 reference strain with MIC values of 4 μg mL⁻¹, and a methicillin-resistant Staphylococcus aureus (MRSA) isolate with MIC values of 8 μg mL⁻¹. It was possible to infer that enantiomer (−)-26 was responsible for the antibacterial activity (MIC 4 μg mL⁻¹) while (+)-26 had no activity. Furthermore, compound (−)-26 was able to impair S. aureus biofilm production and no significant cytotoxicity towards differentiated and non-differentiated SH-SY5Y cells was observed. Compounds 26, 28, and 29 showed a weak antifungal activity against Trichophyton rubrum clinical isolate with MIC 128 μg mL⁻¹ and presented a synergistic effect with fluconazole.

Indolomethyl pyrazino [1,2-b]quinazoline-3,6-diones were prepared using a one-pot multicomponent polycondensation of amino acids and were evaluated for their antimicrobial activity against a panel of nine bacterial strains and five fungal strains.     

Different effects of sulfur amino acids on prolidase and prolinase activity in normal and prolidase-deficient human erythrocytes

BACKGROUND: Prolidase and prolinase activity is known to be enhanced significantly in some diseases. Recently, the effect of amino acids on prolidase and prolinase activity in normal and prolidase-deficient human erythrocytes was investigated. It was reported that both enzymes were enhanced by glycine and alanine in the presence of MnCl(2). METHODS: Erythrocytes were isolated from heparinized blood from normal human and a patient with prolidase deficiency. Effects of various sulfur amino acids on prolidase and prolinase activities against iminodipeptides in the presence of 1 or 0.1 mmol/l MnCl(2) were investigated. RESULTS: Prolinase activity against prolylglycine in normal and prolidase-deficient erythrocyte lysates was inhibited by L-methionine, NAc-L-methionine and D,L-methionine in a concentration-dependent manner, but D-methionine enhanced the activity in low concentrations (0-20 mmol/l). D,L-Homocysteine inhibited the activity more strongly than other sulfur amino acids tested in a concentration-dependent manner. On the other hand, prolidase activity against glycylproline was enhanced by L-methionine, D-methionine, D,L-methionine, D,L-homocysteine thiolactone and D,L-ethionine. The rates of enhancement by these sulfur amino acids were in the following order: D,L-ethionine>D,L-methionine, D-methionine, D,L-homocysteine thiolactone>L-methionine (10 mmol/l). CONCLUSION: The prolinase activity in normal and prolidase-deficient erythrocyte lysates was inhibited by L-methionine, D,L-ethionine and D,L-homocysteine. On the other hand, prolidase activity in their erythrocyte lysates was enhanced by D,L-ethionine, D-methionine and L-methionine. These results indicate the effects of these sulfur amino acids on prolidase and prolinase activities were different.     

STUDIES ON THE NATURE OF RESISTANCE OF GRAM-NEGATIVE BACILLI TO PENICILLIN : ANTAGONISTIC AND ENHANCING EFFECTS OF AMINO ACIDS

The susceptibility of E. coli and Salmonella to penicillin is highest in a basal medium devoid of amino acids. Blood serum in certain concentrations, meat infusion broth, yeast extract, and casein hydrolysate interfere with the penicillin activity. The effect is apparently due to the antagonism of certain amino acids in the materials. Dicarboxyl-monoamino acids (i.e. aspartic, glutamic, and hydroxyglutamic acids and asparagine) cystine, arginine, histidine, and hydroxyproline are capable of suppressing the effect of penicilhn upon Gram-negative organisms. The antagonism of amino acids is not primarily related to their effect upon the rate of bacterial growth. It is suggested from the experiments detailed, that the antipenicillin activity is due to the effect of the amino acids upon bacterial metabolism. Prepassages in media of various concentrations of antagonistic amino acids alter the resistance of E. coli to penicillin. The changes are in inverse relation to the concentration of the antagonists. The antipenicillin activity of amino acids may be reversed significantly by dl-methionine. The substance, however, reverses only incompletely the antagonism of materials of mixed composition; i.e., casein hydrolysate, meat infusion broth, and serum. Upon addition of methionine, methionine sulfoxide, and threonine, there occurs a marked enhancement of penicillin susceptibility of broth cultures of Brucella, Eberthella, Salmonella, and Shigella. The enhancement is apparently due to the ability of this amino acid mixture to reverse effectively the action of the antagonists present in the cultures. Methionine is essential for the enhancement of penicillin susceptibility. Threonine and methionine sulfoxide facilitate the effect of methionine following a reciprocal quantitative relationship.