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PubMed1994

Proline-specific peptidases of Lactobacillus casei subspecies.

This paper describes the specific activities for proline iminopeptidases, x-prolyl dipeptidyl peptidase and post proline endopeptidase, from each of two subspecies of Lactobacillus casei grown in MRS broth and whey media at 37 degrees C, pH 6.0. The histochemical PAGE of soluble extracts from one subspecies (Lactobacillus casei ssp. casei LLG) indicated that the two enzyme activities were due to distinct proteins. Except for a slight increase in x-prolyl dipeptidyl peptidase activity, the activities of proline imino- and endopeptidases of cells grown in whey medium did not vary markedly from those of cells grown in MRS broth. The effect of inhibitor agents and pH on the activities of proline iminopeptidase and x-prolyl dipeptidyl peptidase were investigated. The temperature optima and storage stability under different conditions were also studied for these activities.

Amino Acid Sequence↗
PubMed1976

Purification and properties of proline reductase from Clostridium sticklandii.

Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

Amino Acid Oxidoreductases↗
PubMed1991

High-resolution, real-space imaging of conformational structures of poly-L-proline helixes.

In 1954, Edsall postulated that the imino-acid proline, which is a frequently found constituent of protein molecules, is a key determinant to the three-dimensional architecture of proteins. It not only should play a fundamental role in stabilizing helical structures of polypeptides, it should allow for sharp bends and even for a complete reversal of the direction of a helix looping back on itself. No direct evidence has yet been published to prove this prediction. Using scanning tunneling microscopy, we have presented high-resolution, real-space images of two conformations of poly-L-proline, where one structure clearly exhibits the predicted 180 degrees back-folding behavior. The measured length, 1.89 nm, of the repeating unit cells agrees with available X-ray data for poly-L-proline I with cis-peptide bonds. We further observe aggregated poly-L-proline II, consisting of highly-ordered, periodically and parallel-linked trans-peptide chains which are 2.4 nm apart from each other. Stacking of these aggregates with their orientation rotated by 90 degrees is also observed.

Microscopy, Scanning Tunneling↗
PubMed1989

Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase.

When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol.

Animals↗
PubMed1989

Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa.

An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.

Amino Acids↗
PubMed1988

Proline aminopeptidase activity as a rapid diagnostic test to confirm bacterial vaginosis.

Two biochemical indicators of bacterial vaginosis, proline aminopeptidase activity and gas-liquid chromatographic analysis, were compared. Five hundred women had their vaginal secretions tested for pH, presence of a positive amine test, levels of volatile and nonvolatile short-chain organic acids, and proline aminopeptidase activity. In addition, direct microscopic and Gram stain examinations were performed. Of the 500 women, 349 (70%) had some form of vaginitis. One hundred sixteen were diagnosed as having bacterial vaginosis, and 69 of these (59%) had Mobiluncus sp on either direct microscopic or Gram stain examination. Two hundred thirty-three had either mixed or other forms of vaginitis. One hundred fifty-one patients were normal. The sensitivity of the proline aminopeptidase assay was 83 and 79%, respectively, in patients having bacterial vaginosis with and without Mobiluncus morphotypes. In contrast, gas-liquid chromatography of short-chain organic acids had sensitivities of 71 and 30%, respectively. Specificity of both assays was about 95%. The greater sensitivity of the proline aminopeptidase assay, especially in patients without Mobiluncus morphotypes, proves its superiority.

Aminopeptidases↗
PubMed1984

Resolution of 5-oxo-L-prolinase into a 5-oxo-L-proline-dependent ATPase and a coupling protein.

5-Oxo-L-prolinase catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic cleavage of ATP to ADP and Pi. In the present research, the enzyme present in a strain of Pseudomonas putida isolated from soil by enrichment culture was found to be composed of two protein components. Neither component alone could catalyze the 5-oxoprolinase reaction, but the reaction was effectively catalyzed when they were mixed. One component (A) exhibited 5-oxo-L-proline-dependent ATPase activity indicating that Component A can interact with both ATP and 5-oxo-L-proline. The other component (coupling protein; B) does not exhibit ATPase activity nor is there evidence that it binds 5-oxo-L-proline. The findings are consistent with (but do not prove) the hypothesis that the Component A catalyzes an initial step in the reaction which involves 5-oxoproline and ATP, such as phosphorylation of 5-oxoproline. The coupling protein (B) may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction.

Adenosine Triphosphatases↗
PubMed1983

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline.

We examined the role of pH gradient and membrane potential in dipeptide transport in purified intestinal and renal brush-border membrane vesicles which were predominantly oriented right-side out. With an intravesicular pH of 7.5, changes in extravesicular pH significantly affected the transport of glycyl-L-proline and L-carnosine, and optimal dipeptide transport occurred at an extravesicular pH of 5.5-6.0 in both intestine and kidney. When the extravesicular pH was 5.5, glycyl-L-proline transport was accelerated 2-fold by the presence of an inward proton gradient. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated glycyl-L-proline transport, and the stimulation was observed in the presence and absence of Na+. A carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced H+ diffusion potential (interior-positive) reduced dipeptide transport. It is suggested that glycyl-L-proline and proton(s) are cotransported in intestinal and renal brush-border membrane vesicles, and that the process results in a net transfer of positive charge.

Animals↗
PubMed1994

Identification of the poly-L-proline-binding site on human profilin.

Profilin is a ubiquitous protein that has been implicated in the signaling pathway leading to cytoskeletal rearrangement in cells. An unusual property of profilin is its high binding affinity for poly-L-proline (PLP). This binding property is conserved in the profilins from diverse species with little sequence homology. We have monitored the binding of PLP to profilin by fluorescence and nuclear magnetic resonance spectroscopies. NMR spectroscopy has identified several residues whose amide nitrogen and amide hydrogen chemical shifts are significantly perturbed by binding of PLP. The affected residues are located at various locations throughout profilin's primary structure; however, mapping the location of the affected residues onto the recently determined three-dimensional solution structure of human profilin indicates that the effects of PLP binding are highly localized. Poly-L-proline binds profilin at the hydrophobic interface between profilin's NH2- and COOH-terminal helices and the upper face of its antiparallel beta-sheet. In contrast, residues located on the opposite side of the profilin structure are unaffected. The extent of the potential interaction surface of the PLP-profilin complex suggests that as few as 6 contiguous prolines would be sufficient for binding profilin. Examination of sequence data bases indicates that stretches of prolines of this length and longer occur in numerous regulatory proteins, suggesting that the ability of profilin to bind polyproline may be an important component of its signaling capabilities.

Amino Acid Sequence↗
PubMed1976

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.

Ascorbic Acid↗
PubMed1993

Solution and solid-state circular dichroism analyses of a human salivary proline-rich glycoprotein repeating domain and its subfragments.

Solution- and solid-state c.d. spectra, as well as surface energetics values, were collected for a series of peptides derived from human salivary proline-rich glycoprotein (PRG). The acronyms and sequences for these peptides are as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)-P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2, and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. The presence of stable poly-L-proline II-like 'mini' helices in the solution state was found to be dependent on peptide chain length, pH, salt, and organic solvent type. Other conformational features such as kinks and beta-/gamma-turns were also found in the larger peptides. Solid-state peptide conformations were not necessarily related to their solution-state counterparts. Poly-L-proline II-like 'mini' helices, kinks, and beta-/gamma-turns were similarly found in the various substrate-bound PRG9 peptides. Surface energetics parameters suggested specific orientations for PRG9 peptides and their constituent acids and homopolymers.

Circular Dichroism↗
PubMed1984

Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro.

Concomitant hydroxylation of proline and lysine residues in protocollagen was studied using purified enzymes. The data suggest that prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2-oxoglutarate; oxygen 5-oxidoreductase, EC 1.14.11.4) are competing for the protocollagen substrate, this competition resulting in an inhibition of the lysyl hydroxylase but not of the prolyl 4-hydroxylase reaction. When the same protocollagen was used for these hydroxylases, the affinity of prolyl 4-hydroxylase to the protocollagen substrate was about 2-fold higher than that of lysyl hydroxylase. Hydroxylation of lysine residues in protocollagen had no effect on the affinity of prolyl 4-hydroxylase, whereas hydroxylation of proline residues decreased the affinity of lysyl hydroxylase to one-half of the value determined before the hydroxylation. When enzyme preparations containing different ratios of lysyl hydroxylase activity to prolyl 4-hydroxylase activity were used to hydroxylase protocollagen substrate, it was found that in the case of a low ratio the hydroxylation of lysine residues seemed to proceed only after a short lag period. Accordingly, it seems probable that most proline residues are hydroxylated to 4-hydroxyproline residues before hydroxylation of lysine residues if the prolyl 4-hydroxylase and lysyl hydroxylase are present as free enzymes competing for the same protocollagen substrate.

Animals↗
PubMed2004

Hepatocyte nuclear factors 1alpha and 4alpha control expression of proline oxidase in adult liver.

Adult liver functions are regulated by several hepatocyte nuclear factors (HNFs). HNF4alpha and HNF1alpha are involved in metabolic functions in the liver. The expression of proline oxidase (PO) and proline dehydrogenase was downregulated in the HNF4alpha liver-specific null mice. In addition, the expression of PO was also diminished in the liver derived from HNF1alpha-null mice. The -160 bp proximal promoter region of the PO gene has two HNF4alpha- and HNF1alpha-binding consensus sites. Transactivation, electrophoretic mobility shift and chromatin immunoprecipitation studies revealed that these regions are important for PO promoter activity. These results suggested that HNF4alpha and HNF1alpha regulate proline metabolism in adult liver.

Aging↗
PubMed1986

Proline inhibits N2-fixation in Anabaena 7120.

The uptake and metabolism of proline by Anabaena 7120 is demonstrated. Uptake is linear over the first hour and it appears that the rate of proline oxidation is sufficient to account for growth is suggested by the finding that exogenous proline represses heterocyst differentiation and inhibits nitrogen fixation in Anabaena 7120, without affecting growth rate.

Cyanobacteria↗
PubMed1976

[Study, by an enzymatic method, of the isomers of L-proline (3H-4) and of the effects of preservation].

By use of procollagen proline hydroxylas which catalyses the substitution of the hydrogen in the trans position on carbon 4 with an hydroxyl on L-proline residues of procollagen, we have shown that the L-[3H-4] proline product of C.E.N. Saclay contained 29 per cent of the trans and 71 per cent of the cis form. It was also possible to verify the molecular stability of the isomers and that a small amount of tritium was randomly linked to the whole molecule.

Isotope Labeling↗
PubMed2002

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7.

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.

Amino Acid Sequence↗
PubMed1998

Comparative study of enzymes related to proline metabolism in tepary bean (Phaseolus acutifolius) and common bean (Phaseolus vulgaris) under drought and irrigated conditions, and various urea concentrations.

There are several mechanisms used by plants for survival in adverse environments such as drought, high temperature and salinity. The objective of this study was to evaluate the drought tolerance of tepary bean as a function of biochemical processes linked to isozyme synthesis and changes in enzymatic activity related to proline metabolism. Mature seeds of common beans var. flor de mayo, Phoseolus vulgaris and tepary beans Phaseolus acutifolius were grown under two water conditions (irrigation and drought), and four levels of urea. Vertical electrophoresis and spectrophotometric techniques were used to evaluate protein patterns, glutamate dehydrogenase (GDH), proline oxidase (PO) and pyrroline-5-carboxylate reductase (P5C reductase) enzyme activities. These enzymes were studied because they are directly related to protein synthesis. Electrophoretic patterns showed more proteins in tepary beans than in common beans with limited irrigation. GDH showed only one isozyme, with a molecular weight between 240) to 270 kDa. A decrease in PO activity was observed in common beans under drought stress with a value of 237 micromol/min, in comparison to irrigation conditions of 580 micromol/min. GDH and P5C reductase enzymes have had higher activity in common beans than in tepary beans under water stress. There was a significant difference only in glutamate dehydrogenase enzyme with respect to urea level. The results suggest that drought tolerance of tepary beans is due to biochemical processes related to proline metabolic enzymes.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1982

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.

Animals↗