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PubMed1987

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12.

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

Antibodies, Monoclonal↗
PubMed2000

An L-proline-dependent proton flux is located at the apical membrane level of the eel enterocytes.

This study has demonstrated the existence of an L-proline-dependent (Na independent) proton flux at the apical membrane level of the eel intestinal absorbing cells. Using isolated eel enterocytes and the pH-sensitive fluorescent dye 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF), it was shown that a 20 mM concentration of the imino acid L-proline in the extracellular medium determined an intracellular acidification of approximately 0.28 pH units. However, neither sucrose nor other amino acids were able to significantly acidify the resting intracellular pH. A hyperbolic relationship between extracellular proline concentration and intracellular proton accumulation was observed. Using both isolated brush-border and basolateral membrane vesicles, it was demonstrated that this proline-proton cotransport mechanism was located at the apical membrane level only. In addition, the existence of a coupling mechanism between proline and proton fluxes was demonstrated by the observation that, in brush-border membrane vesicles, the presence of a pH gradient (pH(in) > pH(out)) stimulated the uptake of L-proline.

Amino Acid Transport Systems, Neutral↗
PubMed1979

Ontogeny of amino acid reabsorption in human kidney. Evidence from the homozygous infant with familial renal iminoglycinuria for multiple proline and glycine systems.

Seven infants (two French Canadian, four Ashkenazi Jewish, and one Greek) with massive selective hyperiminoglycinuria (proline, hydroxyproline, and glycine) were detected by urine screening in the second week of life. Follow-up investigations and family studies revealed that each subject had a benign condition, familial renal iminoglycinuria, an autosomal recessive condition. The family studies (Table 1 and Fig. 1) indicate the presence of at least two different mutant alleles segregating in this small group of probands. Hmozygotes of two forms and one genetic compound were identified. Quantitative studies revealed normal concentrations of proline and glycine in plasma (Fig. 2), normal maturation of creatinine clearance (as an index of glomerular filtration rate) (Fig. 3), and elevated renal clearance of proline and glycine (Table 2). Fractional excretion (CAA/CCR) of both proline and glycine in the probands was far in excess of that expected for the normal postnatal infant; FFPro and FEGly approached 100% of the filtered load on occasion (Fig. 4). A schedule of maturing tubular reabsorptive activity was apparent in the proband group. Proline reabsorption matured earlier than glycine reabsorption in the homozygotes (and the genetic compound) as it does in the normal infants (Fig. 5). Our findings suggest that three gene products serve net tubular reabsorption of imino acids and glycine in human kidney. One, affected by mutation in our patients, is responsible for a shared transport activity; a second with preference for proline, and not affected by the mutation, has an "early" schedule of postnatal maturation; and a third with preference for glycine, also not affected by the mutation, has a "late" schedule of maturation.

Amino Acid Metabolism, Inborn Errors↗
PubMed1983

L-Proline transport by isolated renal tubules from newborn and adult rats.

Proline uptake and metabolism has been examined in newborn Sprague-Dawley rat kidney and compared to that in adult animals. [14C]-Proline uptake by renal tubule fragments from newborn rats occurs at the same initial rate as in adult tubules, but at physiologic concentrations achieves significantly higher intracellular levels after 15 min of incubation. Considerable metabolism of the proline taken up was observed in tissue of both ages. Analysis of acid soluble and insoluble tubule fractions from newborn and adults indicates similar degrees of proline incorporation into protein and oxidation to CO2 relative to the amount of radioactivity taken up. A major difference exists, however, with respect to the labeled components of the acid extract: adult tubules convert [14C]-proline to metabolites at a rate twice that of newborn. Analysis of concentration-dependent uptake data reveals two distinct entry systems for proline in both isolated newborn and adult tubules. No difference in the Km or Vmax was found between the young and mature tubules.

Age Factors↗
PubMed2004

L-proline microinjected into the rat ventrolateral medulla induces a depressor response distinct from L-glutamate.

The neurotransmitter candidate L-proline elicits changes in the cardiovascular system via actions in the brainstem. However, its action have not yet been determined in the ventrolateral medulla (VLM), a brain region critical in mediating vasomotor sympathetic nervous system responses. Microinjections of L-glutamate produce depressor responses in the caudal (C) VLM, but pressor responses in the rostral (R) VLM and the caudal pressor area (CPA) in the far caudal CVLM. The present study tested whether microinjections of l-proline in the VLM produce a pattern of hemodynamic responses distinct from that of l- glutamate. Urethane-anesthetized rats received arterial catheters and were implanted with flow probes around the abdominal aorta (supplies hindquarters). The surface of each rat's VLM was then exposed. L-Proline induced dose- dependent depressor responses in the CVLM (0.003-1.0 M, 34 nl), but did not induce hemodynamic responses in sites of the RVLM (0.01-1.0 M, 34 nl) that responded to L-glutamate (0.01 M, 34 nl). L-Proline injections (0.1 M, 34 nl) induced rapid and consistent depressor responses correlated with coincident decreases in hindquarter resistance (arterial blood pressure/flow) in the CVLM and CPA, but only inconsistent responses in a few sites in the RVLM. In summary, L-proline induced a distinct pattern of depressor responses preferentially in caudal regions of the VLM, and these depressor effects were associated with decreases in hindquarter resistance. These findings indicate that L-proline may have unique roles including cardiovascular regulation independently from L-glutamate, especially in caudal region of the VLM, via a mechanism that involves altering hindquarter resistance.

Animals↗
PubMed1983

Effect of proline on arginine uptake and nitrogen metabolism of lactating goats.

The hypothesis that addition of proline may save dietary protein for milk production was tested by administration of proline to two goats through a duodenal cannula. Because proline is synthesized by the mammary gland in vitro from arginine, the effect of proline supplementation on arginine uptake by the gland was tested in vivo. Arginine uptake, calculated from arterio-venous difference, dropped significantly in both animals, especially in the morning 1 h after milking, when a low-protein diet was fed. Milk production and total nitrogen in milk were not affected significantly by proline supplementation. A trend was toward decrease of milk orotic acid and an increase of milk fat due to proline supplementation.

Animals↗
PubMed1975

Serum free proline and free hydroxyproline in patients with chronic liver disease.

Serum free proline and free hydroxyproline were determined in 71 patients with liver disease and in 62 control subjects. The group with liver disease included 60 patients with liver cirrhosis and 11 with chronic active liver disease. Forty-five of the cirrhotic patients were alcoholics, 9 of which were studied during an episode of alcoholic hepatitis. The control group consisted of 24 healthy volunteers, 19 primary malnourished patients, and 19 severely ill patients without liver disease. The values obtained in normal subjects were quite constant; no differences related to sex or age were detected. In patients with malnutrition, and especially in severely ill patients, the proline values were always below the normal limit. Patients with nonalcoholic cirrhosis or chronic active liver disease had serum proline and hydroxyproline values similar to those of normal subjects. However, the patients with alcoholic liver cirrhosis had proline and hydroxyproline values significantly higher than the normal group. Furthermore, in patients with alcoholic hepatitis the serum free proline values were significantly higher than in the other groups. The results suggest that alcohol might have a direct effect on proline metabolism or facilitate its release from the liver cell.

Adolescent↗
PubMed1992

A 13-amino acid n-terminal domain of a basic proline-rich protein is necessary for storage in secretory granules and facilitates exit from the endoplasmic reticulum.

We have investigated the role of different domains of a salivary basic proline-rich protein in intracellular transport and sorting of proline-rich proteins to the secretory granules. We have cloned a full-length cDNA of a basic proline-rich protein from the rat parotid and expressed it in AtT-20 cells. It was correctly sorted into secretory granules as shown by EM immunolocalization and by its presence in 8-bromocyclic AMP-stimulated secretion. Deletion of the N-terminal thirteen amino acid domain upstream from the proline-rich domain eliminated storage whereas deletion of the C-terminal 20-amino acid domain downstream from the proline-rich domain had no effect. Intracellular transport of full-length and mutant proline-rich proteins was unusually slow due to slow exit from the endoplasmic reticulum. However, the rate of transport increased with increasing level of expression for the full-length protein and the C-terminal deletion mutant. In contrast, the rate of transport of the N-terminal deletion mutant was independent of the level of expression. These results imply that the N-terminal domain is necessary for both storage and efficient intracellular transport. Moreover, interactions (self-aggregation?) that mediate sorting may begin as early as the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1991

Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006.

The gene encoding for an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) was sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (1686 base pairs) corresponded to a protein of 562 amino acid residues with a Mr of 66,502. Its predicted amino acid composition, Mr, and N-terminal sequence of 12 residues were consistent with those determined for B. thermoglucosidasius oligo-1,6-glucosidase. The deduced sequence of the enzyme was 72% homologous to that of a thermolabile oligo-1,6-glucosidase (558 residues) from Bacillus cereus ATCC7064 (mesophile). B. cereus oligo-1,6-glucosidase contained 19 prolines. Eighteen of these were conserved at the equivalent positions of B. thermoglucosidasius oligo-1,6-glucosidase. This enzyme contained 14 extra prolines besides the conservative prolines. The majority of extra prolines was replaced by polar or charged residues (Glu, Thr, or Lys) in B. cereus oligo-1,6-glucosidase. The extra prolines were responsible for the difference in thermostability between these two enzymes. We suggested that 11 of the extra prolines in B. thermoglucosidasius oligo-1,6-glucosidase occur in beta-turns or in coils within the loops binding adjacent secondary structures.

Amino Acid Sequence↗
PubMed1990

In vivo cloning of proline genes and its expression in Escherichia coli.

The wild type proA+, B+ genes of E. coli were cloned in vivo using a plasmid containing a mini-Mu replicon, pEG5005. The cloning frequency was about 1.46 x 10(-3)/Kanr transductant. Genetic and biochemical analysis of these clones indicated that the proA+, B+ genes are on the plasmid pEG5005. The secretion of proline were assayed for 500 Pro+ clones. However, no proline accumulation was detected. A Pro+ clone pPR3 was mutagenized in vivo by NTG and the mutants resistant to D-proline were obtained. One of the Dpr mutants pPR7 was found to produce 0.35 mg/ml proline in a proA B deletion strain. When pPR7 was transferred into a proline producing strain, the yield of proline increased up to 2.5 mg/ml, which is 7 and 2.5 times higher than that of the donor and recipient respectively. The physical maps of pEG5005 and pPR3 were roughly established.

Chromosome Mapping↗
PubMed1977

Physiologic interactions between L-proline and D-glucose in Leishmania tarentolae, L. donovani and Trypanosoma scelopori culture forms.

L-proline is more completely catabolized by Leishmania tarentolae and Trypanosoma scelopori than by L. donovani. In the two former organisms the activity of 14CO2 derived from L-proline-14C was as high or higher than that derived from D-glucose-14C; in L. donovani more 14CO2 is derived from D-glucose-14C than from L-proline-14C. Addition of unlabeled D-glucose to cells incubated with L-proline-14C resulted in increased 14C-activity of TCA-soluble fractions of L. tarentolae and T. scelopori; 14C-activity of 5% TCA insoluble fractions of these cells remained unchanged. Some of the increased 14C-label found in the soluble fraction was in glutamate and aspartate. This increased activity in glutamate and aspartate was accompanied by a decrease in alanine-14C in the free amino acid pool. These results suggest that there are complex physiological interactions between glucose and proline in the metabolism of L. tarentolae, L. donovani and T. scelopori. In addition, proline appears to have a more central role in the amino acid metabolism of L. tarentolae and T. scelopori whereas glucose may be more significant in L. donovani.

Aspartate Ammonia-Lyase↗
PubMed1987

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase.

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.

Amino Acid Transport Systems, Neutral↗
PubMed1985

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs.

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.

Amino Acid Sequence↗
PubMed1984

Biosynthesis of salivary proteins in the parotid gland of the subhuman primate, Macaca fascicularis. Cell-free translation of the mRNA for a proline-rich glycoprotein and partial amino acid sequence and processing of its signal peptide.

The major anionic proline-rich proteins in the parotid and submandibular secretions of subhuman primates and man perform the important biological function of inhibiting crystal growth of calcium phosphate salts from saliva, which is supersaturated with calcium phosphate salts, thereby preventing excess deposition of hydroxylapatite on tooth surfaces. The present work was initiated as a first step towards investigating proline-rich protein biosynthesis in parotid glands using the subhuman primate, Macaca fascicularis, as a model system. RNA was isolated from macaque parotid glands and separated into poly(A)-enriched and poly(A)-deficient fractions by chromatography on oligo(dT)-cellulose. The mRNAs in both fractions promoted incorporation of radiolabeled amino acids into polypeptides in an mRNA-dependent reticulocyte lysate translation system. Five major proline-rich polypeptides were detected and one of these was shown to be the in vitro precursor of the major anionic macaque proline-rich protein (MPRP), which is the structural and functional counterpart of the major anionic proline-rich proteins in the parotid and submandibular secretions of man (Oppenheim, F.G., Offner, G.D., and Troxler, R.F. (1982) J. Biol. Chem. 257, 9271-9282). Radiosequencing of the material in anti-MPRP immune precipitates showed that the in vitro precursor of MPRP contained an 18-residue signal peptide. The in vitro precursor of MPRP was processed in dog pancreas vesicles to a form with a lower apparent Mr and with an NH2-terminal amino acid sequence identical to that of native MPRP. The phenylthiohydantoin derivatives of Ala and Ile were detected at residue 9 and those of Val and Met were detected at residue 16 of the signal peptide. This indicated that the in vitro precursor of MPRP, which migrated electrophoretically as a single band in anti-MPRP immune precipitates, contained two different in vitro polypeptides derived from two different mRNAs. These results are discussed in the context of the genetic polymorphism among the major anionic proline-rich proteins in the parotid and submandibular secretions of man.

Animals↗
PubMed1984

N-nitrosation of proline in smokers and nonsmokers.

Endogenous nitrosation of proline was investigated in smokers and nonsmokers. Volunteers consumed a volume of beet juice equivalent to 325 mg nitrate, and 1 hour later they consumed 500 mg proline. In separate experiments volunteers ingested proline alone. Twenty-four-hour urine samples were collected and analyzed for N-nitrosoproline. When proline alone was ingested, there was no significant difference in urinary nitrosoproline excretion between smokers and nonsmokers. When beet juice and proline were consumed, however, smokers produced approximately 2.5 times as much N-nitrosoproline as nonsmokers. Salivary thiocyanate levels were approximately 3.2 times higher in smokers compared to those in nonsmokers. Salivary nitrite levels of smokers and nonsmokers, either before or after beet juice consumption, were not different. Salivary nitrate concentrations, however, were higher in nonsmokers than in smokers after beet juice consumption but not before. Our results suggest that the higher level of salivary thiocyanate in smokers is responsible for the increased rate of endogenous nitrosation of proline in this group compared to the rate in nonsmokers. Nitrosating agents in cigarette smoke do not appear to play a significant role.

Adult↗
PubMed1982

Effect of cell density on the incorporation of [3H] proline into collagen by rabbit aortic smooth muscle cells in culture.

When stationary phase cultures of rabbit aortic smooth muscle cells (SMCs) with different cell densities were incubated with [3H] proline in order to study the rates of collagen and total protein syntheses, the incorporation of [3H] proline into collagen and total protein decreased with increasing cell density. This association was mainly due to a negative correlation between cell density and the specific activity of [3H] proline within the free intracellular proline pool. After correction for the specific radioactivity of free intracellular proline, the synthesis rate of collagen was not significantly correlated with cell density, but the negative association between cell density and the incorporation of [3H] proline into total protein was not completely abolished.

Animals↗
PubMed1995

Proline motifs in peptides and their biological processing.

Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain in that the side-chain is cyclized back onto the backbone amide position. Inside an alpha-helix the possibility of making hydrogen bonds to the preceding turn is lost and a kink will be introduced. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G-protein-coupled receptors, V3 loops of the HIV envelope glycoprotein gp 120, and neuro- and vasoactive peptides. Only a limited number of peptidases are known to be able to hydrolyze proline adjacent bonds. Their activity is influenced by the isomeric state (cis-trans) as well as the position of proline in the peptide chain. The three proline specific metallo-peptidases (aminopeptidase P, carboxypeptidase P and prolidase) are activated by Mn2+, whereas the three serine type peptidases cleaving a post proline bond (prolyl oligopeptidase, dipeptidyl peptidase IV, and prolylcarboxypeptidase) share the sequential order of the catalytic Ser-Asp-His triade, which differentiates them from the chymotrypsin (His-Asp-Ser) and subtilisin (Asp-His-Ser) families. An endo or C terminal Pro-Pro bond and an endo pre-Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme.

Amino Acid Sequence↗
PubMed1996

A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter.

The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.

Amino Acid Transport Systems, Neutral↗