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PubMed1999

Developmental regulation of proline transport in Leishmania donovani.

Leishmania donovani are the causative agents of kala azar in humans. These organisms cycle between the proline-rich environment of the sand fly vector (extracellular promastigotes) and the sugar-rich condition in the mammalian host (intracellular amastigotes). Parasites have adapted to these extreme changes in proline concentrations: promastigotes utilize proline as a carbon source, whereas amastigotes utilize sugars and fatty acids. Previous studies have suggested that promastigotes and amastigotes express distinct proline transporters. However, the information available on these transporters is limited. In this work, proline transport was investigated in axenic L. donovani cultures. Three transport systems were identified: cation-dependent and -independent proline transporters in promastigotes (systems A and B, respectively) and a single cation-independent transporter in amastigotes (system C). Systems A and C have broad specificity to almost all amino acids and obtain optimum activity at acidic pH ranges (pH 6 and 5, respectively). System B is more specific to proline, as it is inhibited by only five amino acids. Temperature response analyses indicated that the transporters of both promastigotes and amastigotes perform best at 37 degrees C. The activity of system A during parasite differentiation was assessed. The transport activity of system A disappeared 3 days after promastigotes were induced to differentiate into amastigotes. In these cells, elevated temperature and acidic pH each suppressed the activity of system A. When amastigotes were induced to differentiate back into promastigotes, system A resumed its activity 24 h after differentiation was initiated. In conclusion, L. donovani obtain proline transport systems that are stage specific, regulated by both pH and temperature. This paper constitutes the first investigation of amino acid transport in axenic L. donovani.

Amino Acid Transport Systems, Neutral↗
PubMed2000

Sequence conservation provides the best prediction of the role of proline residues in p13suc1.

The unique nature of the proline side-chain imposes severe constraints on the polypeptide backbone, and thus it seems likely that it plays a special structural or functional role in the architecture of proteins. We have investigated the role of proline residues in suc1, a member of the cyclin-dependent kinase (cks) family of proteins, whose known function is to bind to and regulate the activity of the major mitotic cdk. The effect on stability of mutation to alanine of all but two of the eight proline residues is correlated with their conservation within the family. The remaining two proline residues are located in the hinge loop between two beta-strands that mediates a domain-swapping process involving exchange of a beta-strand between two monomers to form a dimer pair. Mutation of these proline residues to alanine stabilises the protein. cdk binding is unaffected by these mutations, but dimerisation is altered. We propose, therefore, that the double-proline motif is conserved for the purpose of domain swapping, which suggests that this phenomenon plays a role in the function of cks proteins. Thus, the conservation of the proline residues is a good indicator of their roles in suc1, either in the stabilisation of the native state or in performing functions that are as yet unknown. In addition, the strain resulting from two of the proline residues was relieved successfully by mutation of the preceeding residue to glycine, suggesting a general method for designing more stable proteins.

Amino Acid Motifs↗
PubMed1983

Alanine-resistant mutants of Chinese hamster ovary cells, CHO-K1, producing increases in velocity of proline transport through the A, ASC, and P systems.

We have developed a method for the isolation of transport mutants with increases in velocity of transport through the A and ASC systems and through a newly discovered P system utilizing the amino acid antagonism between A system amino acids and proline in CHO-K1 pro- cells. Mutants alar2 and alar3, isolated in a single-step procedure, resistant to 25 mM alanine in MEM-10 plus 0.05 mM proline are pro-, stable, cross resistant to alpha-(methylamino)isobutyric acid (MeAIB) and show an approximately twofold increase in the initial velocity of proline uptake. Ethyl methane sulfonate (EMS) increases the frequency of pro- alar clones in the population by at least 50 times the spontaneous frequency. The increased velocity of proline transport by alar2 and alar3 can be attributable to the 1.5 to 3 times increase in velocity of transport of proline through systems A, ASC, and P. The Vmax for proline transport through the A system has increased two times for alar2 while the Km and Vmax for alar3 has increased by 1.4 and 2.3 times that of CHO-K1. There is a corresponding increase in Vmax of proline transport by alar2 through the P system. The P system is defined operationally as that portion of the Na+-dependent velocity that remains when the A, ASC, and glutamine-inhibitable fraction are eliminated. The system is concentrative. Proline appears to be the preferred substrate. Li+ cannot be substituted for Na+. The system is moderately dependent upon pH. It obeys Michaelis-Menten kinetics and is not derepressible by starvation. There is no evidence for an N system in CHO-K1.

Alanine↗
PubMed1997

Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants.

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells. In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae. To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S. cerevisiae. When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at -20 degrees C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress. These results suggest that the increased flux in the metabolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts.

Amino Acids↗
PubMed2001

Substrate recognition by proline permease in Salmonella.

Proline transport is required for catabolism of proline as a carbon, nitrogen, and energy source, and for accumulation of proline during adaptation to osmotic stress. These physiological processes are widespread in nature, and play essential roles in the virulence of both prokaryotic and eukaryotic pathogens. In enteric bacteria, the major proline permease is encoded by the putP gene. To identify the structural features required for substrate recognition by PutP, we assayed the transport and toxicity of a variety of natural and synthetic derivatives of proline. The results indicate that the substrate binding site of proline permease consists of a hydrophobic pocket that accommodates C3, C4, and C5 of the pyrrolidine ring. Both 4- and 5-membered rings fit into the substrate binding pocket, but 6-membered rings are excluded. Analogs with substituents on the C4 position are also excluded. In addition, the binding site includes a hydrophilic region that recognizes the imino and carbonyl groups. A free carboxyl group is not required. Taken together, these results may be used to design new synthetic inhibitors of proline transport that can effectively block proline uptake by microbial pathogens.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Modeling proline ligation in the heme-dependent CO sensor, CooA, using small-molecule analogs.

CooA, the only protein known to employ proline as a heme ligand, is a CO-activated transcription factor found in the bacterium Rhodospirillum rubrum. Proline is a heme ligand in both the Fe(III) and Fe(II) states; the sixth ligand is cysteinate in Fe(III) CooA and histidine in Fe(II) CooA. When CO binds to Fe(II) CooA, it selectively replaces the proline ligand, activating the protein. The proposed roles of proline are to stabilize the heme pocket during the redox-mediated ligand switch and to form a weak metal-ligand bond that is preferentially cleaved to bind CO. To explore this latter proposal, binding affinity, structural, and density functional theory computational studies were performed using pyrrolidine and 2-methylpyrrolidine as analogs of proline, and imidazole as an analog of histidine. Measurement of the binding properties of these amino acid analogs in two different protein environments, CooA variant deltaP3R4 and myoglobin, revealed that CooA is tailored to accept the bulky proline ligand. Furthermore, the high pKa of proline facilitates selective replacement by CO. Model metalloporphyrin X-ray and computational structures suggest that the key factor leading to lengthening of the Fe-ligand bond and decreased binding affinity is steric hindrance at the C-2 position of the pyrrolidine ring. These data afford a more complete understanding of how CooA utilizes the weak proline ligand to direct CO to the distal position, thus ensuring selective retention of the histidine ligand.

Bacterial Proteins↗
PubMed2006

Proline induces alterations in nucleotide hydrolysis in rat blood serum.

The main objective of the present study was to evaluate the in vivo (acute and chronic) and in vitro effects of proline on serum nucleotide hydrolysis. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 (micromol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1 h, 3 h or 12 h later. Results showed that acute proline administration provoked a decrease in ATP (42%) and ADP (49%) hydrolysis when rats were sacrificed 1 h after the injection. Furthermore, in rats killed 3 h and 12 h after acute injection, no change in nucleotide hydrolysis were observed. For chronic treatment, buffered proline was injected subcutaneously twice a day at 10 h intervals from the 6(th) to the 28(th) day of age. Rats were sacrificed 3 h or 12 h after the last injection. Chronic administration of proline did not alter the nucleotide hydrolysis when the rats were killed 12 h after the last injection, but decreased ATP (15%) and ADP (32%) hydrolysis when rats were sacrificed 3 h after the last injection. The in vitro effect of proline (3.0 microM - 1.0 mM) on serum nucleotide hydrolysis was also investigated; results showed that 1.0 mM of proline significantly increased ATP (45%), ADP (55%) and AMP (49%) hydrolysis. The data indicate that proline in vivo and in vitro alters nucleotide hydrolysis, which may be involved in the pathogeny of hyperprolinemic patients.

Animals↗
PubMed1979

An inducible proline transport system in Candida albicans.

1. When Candida albicans cells were preincubated with proline or grown in the presence of proline as the sole nitrogen source they exhibited a rapid increase in the influx of proline (the inducible transport system). 2. The induction appeared to be specific for proline and also demonstrated in other Candida species. 3. Both the inducible and constitutive proline uptake systems exhibited similar characteristic features. 4. The nature of the inducer for proline uptake in C. albicans appeared to be free proline. 5. The development of the inducible proline transport system was dependent on concomitant synthesis of RNA and protein and the induction was not affected by glucose or any other carbon sources used.

Amino Acids↗
PubMed1992

Sodium ion and proline binding sites in the Na+/proline symport carrier of Escherichia coli.

Proline binding activity of the Escherichia coli Na+/proline symport carrier is inhibited by a sulfhydryl reagent, N-ethylmaleimide (NEM). Proline and its analogs protected the carrier against the NEM-inactivation in a Na+ (or Li+)-dependent manner. Na+ alone, even in the absence of proline, partially protected it from the NEM-inactivation. Mutant proline carriers, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively (Yamato, I. and Anraku, Y. (1988) J. Biol. Chem. 263, 16055-16057) were also analyzed for cation-dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. Furthermore, the proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. These results indicate that Cys-344, which is located in the putative eighth membrane-spanning domain in the carrier, is a cysteine residue functionally involved in the high-affinity binding for sodium ion and proline.

Amino Acid Sequence↗
PubMed1985

Transport of glycyl-L-proline by human intestinal brush border membrane vesicles.

This study characterizes the transport of [1-14C]glycyl-L-proline into purified brush border membrane vesicles prepared from human small intestine. Time-course uptake curves of glycyl-L-proline were similar under sodium thiocyanate or potassium thiocyanate gradient conditions (extravesicular greater than intravesicular) and did not show any overshoot phenomena. The transport of glycine and proline, however, was stimulated by the presence of sodium gradient. Measurement of peptide uptake with increasing medium osmolarity showed that glycyl-L-proline was transported into an osmotically reactive intravesicular space with insignificant binding to the surface of the vesicles. Only 2% of the glycyl-L-proline in the incubation media was hydrolyzed after 10 min of incubation. Also, there was no hydrolysis of peptide transported into the intravesicular space. The effects of increasing concentrations of glycyl-L-proline on uptake showed that uptake of the peptide was saturable and conformed to Michaelis-Menten kinetics with a Km of 4.1 +/- 0.5 mM and a Vmax of 1.53 +/- 0.07 nmol/mg protein X 0.5 min. Free amino acids did not inhibit the transport of glycyl-L-proline while dipeptides and tripeptides exerted appreciable inhibition (up to 60%). Our results show that human small intestinal brush border membrane vesicles transport glycyl-L-proline as an intact peptide by a carrier-mediated, Na+-independent process.

Amino Acids↗
PubMed1985

Hormonal effects on L-proline transport in coho salmon (Oncorhynchus kisutch) intestine.

The hormonal control of proline transport in pyloric ceca was studied in regard to the effects of cortisol, growth hormone (GH), epinephrine, and 3-isobutyl-1-methylxanthine (IBMX). Cortisol pellets implanted in yearling freshwater (FW) salmon for 2 weeks elevated plasma cortisol levels six times above that of control fish. The maximal influx (Jmax) and the half-saturation constant (Kt) of proline influx were twofold greater in cortisol-treated fish than the values in controls; the apparent passive permeability coefficient (Pa) was significantly reduced in the former group. FW salmon implanted with GH for 2 weeks showed increased body weight gain and a higher Jmax of proline influx compared with that of control fish. GH treatment resulted in a higher Pa of proline influx as well as in a 30% increase in area-specific intestinal dry weight. Thus, GH and cortisol may play a regulatory role in intestinal amino acid absorption during salmon development. The in vitro effects of epinephrine and the phosphodiesterase inhibitor, IBMX, on short-circuit current (Isc) and proline influx in salmon intestine were examined. Epinephrine (10(-6) M) caused a rapid increase in negative Isc (mucosa, ground). Pyloric ceca preincubated with epinephrine for 30 min showed reduced total proline influx compared with influx in paired control tissues. Epinephrine increased and IBMX decreased the Kt of proline influx; IBMX also reduced Jmax. The possible interaction between the effects of epinephrine and IBMX on ion transport and Na+-coupled proline influx are discussed.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling.

Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 mumol/kg for alanine, 578 mumol/kg for glutamate and 296 mumol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline.

Adrenalectomy↗
PubMed1981

Proline biosynthesis by Fasciola hepatica at different developmental stages in vivo and vitro.

The ability of Fasciola hepatica to produce proline was assessed by measuring activity of the proline biosynthesis enzyme ornithine-delta-transaminase, and the arginine-dependent production of proline in vitro. This was related to the changes in bile proline of rats during the course of infection. It was found that the total production of proline and total enzyme activity per fluke increased as the fluke increased in size. However, while the specific enzyme activity remained constant throughout development, there was a preferential increase in proline production relative to protein content of the flukes just prior to their migration into the bile ducts. This early amplification of proline production could provide the stimulus to induce hyperplasia in the bile duct in preparation for fluke entry as proline production relative to protein content of fluke dropped markedly after migration into the bile ducts and never again reached the pre-migration level.

Animals↗
PubMed1978

Unusual solution properties of proline and its interaction with proteins.

Proline in aqueous solution shows several properties which are unusual for low molecular weight substances. Investigations of solubility, density and viscosity revealed behaviour which is characteristic for hydrophilic colloids. 1H-NMR studies indicated a strong hydrogen bonding of water in proline solutions, especially at high concentrations of the solute. From these results it was concluded that proline forms aggregates by stepwise stacking and hydrophobic interaction of the pyrrolidine ring. Thus, the proposed multimer contans a hydrophobic backbone and hydrophilic groups on the surface, exposed to water. Proline solutions are able to increase the solubility of sparingly soluble proteins. The enhancement effect depends on the nature of the protein and on the proline concentration. It is postulated that by a hydrophobic interaction of proline with hydrophobic surface residues of proteins their hydrophilic area is increased. The presence of proline in solutions of the well soluble protein bovine albumin reduces the precipitation of this protein by ethanol and (NH4)2SO4, presumably by an increased water-binding capacity of the proline-protein solution.

Chemical Phenomena↗
PubMed1984

Proline and hepatic lipogenesis.

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.

Animals↗
PubMed2006

Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities.

Proline and betaine accumulate in plant cells under environmental stresses including salt stress. Here, we investigated effects of proline and betaine on the growth and activities of antioxidant enzymes in tobacco Bright Yellow-2 (BY-2) culture cells in suspension under salt stress. Both proline and betaine mitigated the inhibition of growth of BY-2 cells under salt stress and the mitigating effect of proline was more than that of betaine. Salt stress significantly decreased the activities of superoxide dismutase (SOD), catalase and peroxidase in BY-2 cells. Exogenous application of proline or betaine alleviated the reduction in catalase and peroxidase activities but not SOD activity under salt stress. In addition, proline was found to be effective in alleviating the inhibition of salt stress-induced catalase and peroxidase activities in BY-2 cells. Neither proline nor betaine directly scavenged superoxide (O(2)(-)) or hydrogen peroxide (H(2)O(2)). It is concluded that exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine because of its superior ability to increase the activities of antioxidant enzymes.

Antioxidants↗
PubMed1997

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

Alamethicin↗
PubMed2002

Beetles' choice--proline for energy output: control by AKHs.

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.

Animals↗