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PubMed1997

Role of the pyrrolidine ring of proline in determining the substrate specificity of cdc2 kinase or cdk5.

To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser55-Pro56-Gly-Gly58-Ala-Tyr-NH2], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the K(m) value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the K(m) value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the K(m) value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a beta-turn at the phosphorylation site and that the K(m) values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high V(max) value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.

CDC2 Protein Kinase↗
PubMed1999

Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of delta1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC.

Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions. Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate. Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase. Both enzymes were present in crude extracts of C. sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected. PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200. The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence. Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively. The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0). Studies with growing cells of C. sticklandii and [15N]proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction. The proC gene encoding PCA reductase of C. sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli. The enzyme exhibited high homologies to PCA reductases from different sources. Thus, C. sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

Amino Acid Sequence↗
PubMed1982

s-Cis...and s-trans isomerism in acylproline analogs. Models for conformationally locked proline peptides.

N-Acetyl-2,3-dehydroproline, N-acetyl-5-oxo-L-proline, N-acrylyl-L-proline, N-acetyl-L-azetidine-2-carboxylic acid and N-acetyl-D,L-pipecolic acid have been examined in 2H2O by 1H and 13Cn.m.r. for the purpose of finding s-cis or s-trans locked acylprolines. Conformationally locked acylprolines could be incorporated into proline-containing peptide hormones such as angiotensin and thyroliberin in order to determine the rotational state of the peptide bond to proline in the hormone receptor complex. The populations of trans and cis rotational isomers were determined as a function of p2H in order to assign the trans and cis isomers and to compare the populations in all the acylprolines at neutral p2H, where the cis isomer is normally present. Proton spectra were also recorded at from 7 degrees to 75 degrees in order to qualitatively determine the exchange rate between the isomers. The majority of these analogs exhibit a cis-trans isomerization similar to that of N-acetyl-L-proline in the ratio of trans to cis rotational isomer found at neutral p2H (about 1:1), the temperature dependence of the population ratio (none), and the coalescence temperature for proton resonances (greater than 75 degrees). However, N-acetyl-5-oxo-L-proline was found to be greater than 98% s-trans at neutral pH, compared to 50% s-trans in N-acetyl-L-proline, and therefore a good candidate for synthesis of an s-trans locked peptide hormone. N-Acetyl-2,3-dehydroproline rapidly exchanges between s-cis and s-trans in contrast to all other proline analogs examined and exhibits coalescence of the beta-proton cis and trans resonances at 45 degrees. Titration with the shift reagent Pr+++ was employed to confirm the assignments of the cis and trans methyl resonances of all of the N-acetyl compounds except N-acetyl-5-oxo-L-proline.

Magnetic Resonance Spectroscopy↗
PubMed1995

Functional consequences of proline mutations in the putative transmembrane segments 6 and 10 of the glucose transporter GLUT1.

Proline residues are thought to play a characteristic structural and/or dynamic role in various membrane proteins [Williams, K.A. & Deber, C.M. (1991) Biochemistry 30, 8919-8923]. By use of site-directed mutagenesis and functional expression of mutant glucose transporters in Xenopus oocytes, we investigated the effects of single proline substitutions in the putative helices 6 and 10 on GLUT1-mediated glucose transport. Proline residues of helix 6, that are conserved in all human glucose-transporter isoforms except for the human GLUT2, were mutated either to alanine or to the corresponding residues of GLUT2, i.e. to histidine (P187H), arginine (P196R) or phenylalanine (P205F). In addition, the three proline amino acids within the domain G382-P-G-P-I-P of helix 10 were individually replaced with either alanine or glutamine residues. In all cases, transport function was retained when each individual proline residue was replaced with alanine. Substitution of proline 196 by arginine (P196R), however, resulted in reduction of 2-deoxy-D-glucose uptake rates by approximately 70%. Since the amount of this mutant transporter protein in plasma membrane and total membrane preparations was found to be decreased, as detected by immunoblotting, no single proline residue seemed to play a critical role in maintaining the catalytic activity of GLUT1. However, structural changes introduced by incorporation of the neutral polar amino acid glutamine at each single proline position of helix 10 almost completely abolished 2-deoxy-D-glucose uptake. Thus, the specific chemical structure of the side chain of the substituted amino acid rather than the unique property of proline residues for cis-trans isomerization seemed to determine the consequences on glucose transport.

Animals↗
PubMed1987

Sodium-dependent proline uptake in the rat hippocampal formation: association with ipsilateral-commissural projections of CA3 pyramidal cells.

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.

Animals↗
PubMed1981

L-proline transport by brush border membrane vesicles prepared from human placenta.

1. Brush border microvillous plasma membranes were prepared from syncytiotrophoblast of human term placenta by a method of differential centrifugation. 2. Such plasma membranes form closed, osmotically active, right-side-out vesicles into which L-proline (188 microM) is shown to be transported in a time-dependent manner. There is no detectable metabolism of L-proline within the vesicles during 30 min of incubation. 3. Transient accumulation of L-proline to levels of up to three times its equilibrium value occurs in the presence of an inward gradient of sodium chloride. The proline and sodium are shown to have reached electrochemical equilibrium by 30 min, at which stage there is about 100 pmol L-proline mg protein-1. 4. This transient accumulation is abolished by the prior equilibration of the sodium chloride gradient, or by the replacement of sodium by inwardly directed gradients of other cations. Entry of the amino acid into the vesicles is also shown to be influenced by the permeability of the anion in the medium and by an imposed potassium diffusion potential. L-Proline transport across the brush border membrane of human placental syncytiotrophoblast is thus a sodium-dependent, electrogenic process. 5. Studies of the transport processes indicate saturation at higher concentrations of L-proline with a 'Km' of about 1 mM; Vmax averaged about 2 nmol mg protein-1 min-1 varied considerably between preparations. 6. L-Proline (188 microM) transport is inhibited competitively by the presence of many amino acids and by the dipeptide glycyl-L-proline. The Ki for inhibition by methyl AIB is 300 microM. 7. These findings are discussed in relation to the mechanism of transplacental amino acid transfer.

Amino Acids↗
PubMed1976

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.

Adenosine Triphosphate↗
PubMed1992

Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12.

The amounts of cytoplasmic water and of all osmotically significant cytoplasmic solutes were determined for Escherichia coli K-12 grown in 3-(N-morpholino)propane sulfonate (MOPS)-buffered glucose-minimal medium containing 0.5 M NaCl in the presence and absence of the osmoprotectants betaine and proline. The goal of this work is to correlate the effects of osmoprotectants on the composition of the cytoplasm with their ability to increase the growth rate of osmotically stressed cells. At a concentration of 1 mM in the growth medium, betaine increases the growth rate more than does proline; choline, which is converted to betaine by E. coli, appears to have an intermediate effect on growth rate. The accumulation of either betaine or proline reduces the cytoplasmic amounts of K+, glutamate, trehalose, and MOPS (the major cytoplasmic osmolytes accumulated in the absence of osmoprotectants), so that at this external osmolarity the total amount of cytoplasmic solutes is essentially the same in the presence or absence of either osmoprotectant. More betaine than proline is accumulated, so the extent of replacement of cytoplasmic solutes is greater for betaine than for proline. Accumulation of these osmoprotectants is accompanied by a large (20 to 50%) increase in the volume of cytoplasmic water per unit of cell dry weight (Vcyto). This effect, which appears to result from an increase in the volume of free water, Vf (as opposed to water of hydration, or bound water), is greater for betaine than for proline. Taken together, these results indicate that the molar effects of betaine and proline on water activity and on the osmotic pressure of the cytoplasm must be significantly larger than those of the solutes they replace. Cayley et al. (S. Cayley, B. A. Lewis, H. J. Guttman, and M. T. Record, Jr., J. Mol. Biol. 222:281-300, 1991) observed that, in cells grown in the absence of osmoprotectants, both growth rate and Vcyto decreased, whereas the amount of cytoplasmic K+ (nK+) increased, with increasing external osmolarity. We predicted that the observed changes in nK+ and Vcyto would have large and approximately compensating effects on key protein-nucleic acid interactions of gene expression, and we proposed that Vf was the fundamental determinant of growth rate in osmotically stressed cells. The properties of cells cultured in the presence of betaine and proline appear completely consistent with our previous work and proposals. Accumulation of betaine and, to a lesser extent, proline shifts the set of linked physiological parameters (nK+, Vcyto, growth rate) to those characteristic of growth at lower osmolarity in the absence of osmoprotectants. Models for the thermodynamic basis and physiological consequences of the effect of osmoprotectants on Vcyto and Vf are discussed.

Betaine↗
PubMed1986

Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts.

Compartmentation of the amino acid precursor pools for protein synthesis in cultured cells can substantially complicate measurements of synthesis rates. This is particularly true for nonessential amino acids such as proline, an amino acid often used in isotopic form to measure collagen synthesis. We have made a comparative study of this problem in cultured IMR-90 fibroblasts using isotopic proline and leucine to measure total protein and collagen synthesis. 3H-leucine in the extracellular (EC) medium equilibrates with tRNA-leucine at an EC concentration of 0.4 mM in both dividing and stationary cells. Thus, under these experimental conditions there is no complicating compartmentation of leucine for protein synthesis. Equilibration of EC and tRNA-bound 3H-proline, however, does not occur even when the EC concentration is in the mM range, based upon simultaneous measurements of synthesis rates using 3H-proline and 3H-leucine together. Furthermore, significant changes in EC proline concentration and specific activity occur over short time intervals (2 hr) if the initial EC proline concentration is below 0.2 mM. Thus, the use of isotopic proline to measure protein synthesis introduces substantial interpretive problems. Serum deprivation causes changes in both total collagen synthesis and the percent of protein synthesis devoted to collagen when measured with either 14C-leucine or 3H-proline. At the same time, isotopic proline remains the better choice for measuring percent collagen synthesis.

Carbon Radioisotopes↗
PubMed1995

Mitigating the effects of halofuginone on skin strength by feeding L-proline to broiler chickens.

Two experiments were conducted to determine whether supplemental levels of L-proline in the diets of broiler chickens would mitigate the skin weakening effect caused by continuous feeding of the anticoccidial halofuginone. In Experiment 1, tensile strength and collagen levels in thigh apteria skin were determined at 21 and 42 d of age in male broilers fed 0, .5, and 1% L-proline with either halofuginone (3 mg/kg) or salinomycin (61 mg/kg). In Experiment 2, the same measurements were made on female broilers receiving diets containing halofuginone and supplemented with 0, .5, or 1% L-proline, 1% L-proline through 21 d of age, or 1% L-glutamic acid through 21 d of age, or a diet containing high L-proline feedstuffs (corn gluten meal and ring dried blood meal). In Experiment 1, dermis thickness of thigh apteria was measured in the males at Day 21. Skin strength was increased in male and female broilers fed halofuginone with addition of .5 and 1% L-proline, respectively, at 21 and 42 d of age. Continuous incorporation of synthetic L-proline into diets was shown to improve skin strength in females, whereas diets formulated to contain high levels of L-proline from feedstuffs, 21-d feeding of L-proline, or L-glutamic acid did not increase skin strength.

Animals↗
PubMed2000

Probing the role of a conserved M1 proline residue in 5-hydroxytryptamine(3) receptor gating.

A conserved proline residue is found in the first transmembrane domain (M1) of every subunit in the ligand-gated ion channel superfamily. The position of this proline between the N-terminal extracellular agonist binding and the second transmembrane (M2) channel lining domains in the primary sequence suggests its possible involvement in the gating of the receptor. Replacing this proline with alanine, glycine, or leucine in the 5-hydroxytryptamine (5-HT)(3A) homomeric receptors expressed in Xenopus laevis oocytes resulted in the absence of 5-HT-induced whole-cell currents, although there were normal levels of specific surface [(3)H]granisetron ([(3)H]BRL-43694) binding sites. To determine what properties of the conserved proline are critical for the function of the channel, two imino acids and an alpha-hydroxy acid were incorporated at the proline position using the nonsense suppression method. trans-3-Methyl-proline, pipecolic acid, and leucic acid were able to replace the conserved proline to produce active channels with EC(50) values similar to that for the wild-type receptor. These trends are preserved in the heteromeric receptors consisting of 5-HT(3A) and 5-HT(3B) subunits in oocytes. The prominent common feature among these residues and proline is the lack of hydrogen bond donor activity, potentially resulting in a flexible secondary structure in the M1 region. Thus, lack of hydrogen bond donor activity may be a key element in channel gating and may explain the high degree of conservation of this M1 proline.

Animals↗
PubMed1992

Glyconeogenesis from L-proline involves metabolite inhibition of the glucose-6-phosphatase system.

L-Proline's glycogenic action is unlike that of other amino acids in that it produces effects beyond those explainable by a simple increase in osmolarity (Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M., and Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959). We postulate that this effect may relate to inhibition of hepatic glucose-6-P hydrolysis by a proline-derived metabolite. We tested this hypothesis with isolated livers from rats fasted 48 h which were perfused with L-proline or L-glutamine. Net glucose and net glycogen production and levels of glucose-6-P and certain other hepatic metabolites were measured. The data obtained support our hypothesis by demonstrating fundamental differences in the metabolic fates of proline and glutamine in the liver. Both pass through alpha-ketoglutarate in the initial stage of gluconeogenesis, but proline supports hepatic glycogen formation while glutamine does not. The concomitant increase in hepatic glucose-6-P and proline-associated glyconeogenesis suggests that inhibition of glucose-6-P hydrolysis by a proline-derived metabolite may divert glucose-6-P produced from proline from glucose production and to glycogen synthesis. This conclusion is supported by the effects of perfusions with and without proline (3-mercaptopicolinate present) on (a) glyconeogenesis and glucose formation from dihydroxyacetone, (b) net glucose uptake and glycogen formation with 30 mM glucose as substrate, and (c) glucose production from endogenous glycogen in perfused livers from fed rats.

Animals↗
PubMed1988

Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12.

Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.

Amino Acid Transport Systems, Neutral↗
PubMed1986

Solubilization and functional reconstitution of the proline transport system of Escherichia coli.

The membrane carrier for L-proline (product of the putP gene) of Escherichia coli K12 was solubilized and functionally reconstituted with E. coli phospholipid by the cholate dilution method. The counterflow activity of the reconstituted system was studied by preloading the proteoliposomes with either L-proline or the proline analogues: L-azetidine-2-carboxylate or 3,4-dehydro-L-proline. The dilution of such preloaded proteoliposomes into a buffer containing [3H]proline resulted in the accumulation of this amino acid against a considerable concentration gradient. A second driving force for proline accumulation was an electrochemical potential difference for Na+ across the membrane. More than a 10-fold accumulation was seen with a sodium electrochemical gradient while no accumulation was found with proton motive force alone. The optimal pH for the L-proline carrier activities for both counterflow and sodium gradient-driven uptake was between pH 6.0 and 7.0. The stoichiometry of the co-transport system was approximately one Na+ for one proline. The effect of different phospholipids on the proline transport activity of the reconstituted carrier was also studied. Both phosphatidylethanolamine and phosphatidylglycerol stimulate the carrier activity while phosphatidylcholine and cardiolipin were almost inactive.

Amino Acid Transport Systems, Neutral↗
PubMed1987

Kinetics of the intestinal brush border proline (Imino) carrier.

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.

Amino Acid Transport Systems, Neutral↗
PubMed1979

Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.

Aminohydrolases↗
PubMed1966

Changes in Amino Acid Content of Excised Leaves During Incubation. III. Role of Sugar in the Accumulation of Proline in Wilted Leaves.

A previously reported accumulation of proline in wilted turnip leaves has been observed in 6 additional species representing 5 different families. The results of experiments on the conditions affecting the behavior of proline in wilted leaves were interpreted to mean that the supply and metabolism of available carbohydrate was essential for proline accumulation.The role of sugar in proline accumulation was determined by incubating wilted leaves with different levels of carbohydrate (produced either by variations in conditions of preillumination or by infiltration with sucrose). The results showed that proline accumulation was greater and most prolonged in wilted leaves with higher sugar and starch contents. No proline accumulation was observed in unwilted leaves. Also some inhibitors of glycolysis and of the tricarboxylic acid cycle prevented proline accumulation in wilted leaves.Based on these results, the interpretation of the role of sugar in proline accumulation is that the oxidation of sugars furnishes alpha-ketoglutarate and NAD(P)H for proline synthesis in wilted leaves.

Journal Article↗
PubMed1977

Inhibition of proline oxidation by water stress.

The conversion of proline to glutamic acid and hence to other soluble compounds (proline oxidation) proceeds readily in turgid barley (Hordeum vulgare) leaves and is stimulated by higher concentrations of proline. This suggests that proline oxidation could function as a control mechanism for maintaining low cellular levels of proline in turgid tissue. In water-stressed tissue, however, proline oxidation is reduced to negligible rates. These results are consistent with the idea that proline accumulation results from inactivation by water stress of normal control mechanisms. It seems likely that inhibition of proline oxidation is necessary in maintaining the high levels of proline found in stressed barley leaves.

Journal Article↗