Nuclear localization of the PEP protein tyrosine phosphatase.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.     

The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Isolation and characterization of human immunodeficiency virus type 1 variants infectious to brain-derived cells: detection of common point mutations in the V3 region of the env gene of the variants.

T-cell-line-tropic human immunodeficiency virus type 1 cannot infect CD4-positive, brain-derived cells. We isolated several new variants that readily infected brain-derived cells. Mutation of proline to serine, to alanine, or to threonine in the well-conserved GPGR sequence in the V3 region of the envelope glycoprotein was found in all these variants. This indicates the importance of amino acid sequences at the tip of the V3 region for brain cell tropism of human immunodeficiency virus type 1.     

Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase.

Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonuclease I produces staggered cuts in the DNA of chromatin

The relationship of cuts made by deoxyribonuclease I (DNase I, EC. 3.1.4.5) on the two strands of DNA of chromatin has been investigated. DNA was extracted from a DNase I digest of rat liver nuclei and incubated with the large fragment of DNA polyrnerase I. Analysis of the products of this incubation indicates the cuts made by DNase I on opposite strands are staggered with respect to one another. A cut on one strand is about two bases in the 3′ direction or eight bases in the 5′ direction from the position on its own strand which is directly across from the cut on the other strand. A different result is obtained when a DNase I digest of native DNA is analyzed. Current models for the organization of DNA in the nucleosome are discussed with respect to these results.     

Identification of the phosphorylation sites in the survival motor neuron protein by protein kinase A

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.     

Validation of the reliability of computational O-GlcNAc prediction

O-GlcNAcylation is an inducible, highly dynamic and reversible posttranslational modification, which regulates numerous cellular processes such as gene expression, translation, immune reactions, protein degradation, protein–protein interaction, apoptosis, and signal transduction. In contrast to N-linked glycosylation, O-GlcNAcylation does not display a strict amino acid consensus sequence, although serine or threonine residues flanked by proline and valine are preferred sites of O-GlcNAcylation. Based on this information, computational prediction tools of O-GlcNAc sites have been developed. Here, we retrospectively assessed the performance of two available O-GlcNAc prediction programs YinOYang 1.2 server and OGlcNAcScan by comparing their predictions for recently discovered experimentally validated O-GlcNAc sites. Both prediction programs efficiently identified O-GlcNAc sites situated in an environment resembling the consensus sequence P-P-V-[ST]-T-A. However, both prediction programs revealed numerous false negative O-GlcNAc predictions when the site of modification was located in an amino acid sequence differing from the known consensus sequence. By searching for a common sequence motif, we found that O-GlcNAcylation of nucleocytoplasmic proteins preferably occurs at serine and threonine residues flanked downstream by proline and valine and upstream by one to two alanines followed by a stretch of serine and threonine residues. However, O-GlcNAcylation of proteins located in the mitochondria or in the secretory lumen occurs at different sites and does not follow a distinct consensus sequence. Thus, our study indicates the limitations of the presently available computational prediction methods for O-GlcNAc sites and suggests that experimental validation is mandatory. Continuously update and further development of available databases will be the key to improve the performance of O-GlcNAc site prediction.     

Effect of variations in salinity and alkalinity of applied irrigation waters on the amino acid make-up of Phaseolus aureus

Summary A marked decrease in the content of various amino acids was observed when the boron concentration was increased from 0.5 to 2.0 ppm particularly at low value of SAR and low level of conductivity. At medium level of conductivity alongwith low level of boron increase in value of SAR definitely suppressed the content of lysine, arginine plus histidine, aspartic acid plus glutamine, threonine plus alanine, proline and cystine in plants. Increase in levels of conductivity specially at low value of SAR and low level of boron substantially affected the content of lysine, arginine plus histidine, aspartic acid plus glutamine, threonine plus alanine, proline and cystine.     

The role of proline in thidiazuron-induced somatic embryogenesis of peanut

Summary Peanut seeds germinated on media supplemented with thidiazuron [TDZ: N-phenyl-N′-(1,2,3-thiadiazol-yl)urea], formed somatic embryos at the hypocotyledonary notch region by Day 35 of the culture period. Supplementation of the culture media with proline, thioproline, or glutamine reduced the total number of embryos formed, but the resulting embryos were larger, greener and had a more synchronous development than the regenerants formed on media containing TDZ alone. Analysis of the endogenous amino acid content of the germinating seeds during the induction phase of somatic embryogenesis revealed accumulation of proline to 6% of the dry seed weight. Concurrent with the emergence of the radicle, the proline concentration remained significantly elevated throughout the expression phase of embryogenesis. Several other amino acids including alanine, aspartate, asparagine, glutamate, glutamine, γ-aminobutyrate (GABA), hydroxyproline, isoleucine, threonine and valine accumulated to peak values approximately 10-fold higher than those of the controls. These results indicate that proline plays a key role in directing the route of TDZ-induced somatic embryogenesis and that TDZ effectively stimulates a cascade of metabolic events resulting in the production of specific metabolites, including amino acids, required for the regenerative process.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.     

Resistance of proline-containing peptides to ruminal degradation in vitro.

Mixed ruminal bacteria utilized an enzymatic digest of casein at a rate faster than that for an enzymatic digest of gelatin, but neither amino acid source was completely utilized even when the incubation period was as long as 96 h. Since the reaction of ninhydrin with the residual nonammonia, nonprotein nitrogen was more than twofold stronger when the samples were hydrolyzed with 6 N HCl, it appeared that much of the residual nitrogen was from peptides. Approximately 66% of the nonammonia, nonprotein, ninhydrin-reactive material could not be recovered as amino acids, but there was a significant decrease in total amino acid nitrogen when the samples were pretreated with a C18 Sep-Pak column to remove peptides. The resistant peptides had an abundance of proline, and subsequent incubations showed that synthetic dipeptides which contained proline were hydrolyzed slowly. Lysine appears to be the amino acid which is most apt to limit ruminant production. Dipeptides containing proline and lysine were hydrolyzed at least fivefold slower than lysine-alanine. Methionine, another potentially limiting amino acid, was also degraded at a slower (2.5-fold) rate when it was present as part of a proline dipeptide.     

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.     

Functional tyrosyl residues of carboxypeptidase A. The effect of protein structure on the reactivity of tyrosine-198

Coupling of bovine carboxypeptidase A with diazotized 5-amino-1H-tetrazole increases esterase activity, decreases peptidase activity slightly, and modifies one tyrosyl residue. Subsequent nitration of the azoenzyme has no further effect on esterase activity, decreases peptidase activity markedly, and modifies a second tyrosyl residue. Analysis of the azopeptides isolated from a chymotrypsin digest of the doubly modified enzyme by affinity, ion exchange, and high pressure liquid chromatography indicates that the principal residue modified by diazo-1H-tetrazole is Tyr-248. Analysis of the nitropeptides isolated by similar procedures indicates that nitration occurs mainly at Tyr-198. This residue becomes susceptible to modification only as a consequence of a conformational change that accompanies azo coupling of Tyr-248. These results describe a unique example of the influence of protein structure on the reactivity of functional amino acid residues and illustrate an important aspect of chemical modification of enzymes.     

Identification of the apparently essential lysine residues in phospholipase C (Bacillus cereus).

Phospholipase C (Bacillus cereus) contains two apparently essential and very reactive lysine residues that may be labelled selectively by pyridoxal 5'-phosphate [Aurebekk & Little (1977) Biochem, J. 161, 159--165]. One of these lysine residues was found in the 25-amino acid N-terminal fragment liberated by CNBr digestion of the pyridoxal-labelled enzyme and identified as lysine-6. Two of the labelled peptides isolated from the chymotryptic digest of pyridoxal-labelled enzyme contained proline, suggesting that the other labelled lysine residue is situated in the same region of the primary structure as the single proline residue of the enzyme.     

Influence de quelques acides aminés libres de l'ovule sur la croissance et le développement cellulaire in vitro du tube pollinique chez Juniperus communis (Cupressaceae)

Influence of some free amino acids of the ovule on growth and cellular development of the pollen tube of Juniperus communis L. in vitro. The extraction and analysis of free amino acids show that a partial complementary relationship exists between the amino acids of the pollen and those of the ovule of Juniperus communis. The main free amino acids of the pollen are threonine, proline, arginine and γ-aminobutyric acid; those of the ovule are threonine, serine, alanine, citrulline and glycine. The addition of the main amino acids of the ovule in the pollen culture media increased the growth and the cell development of pollen tubes cultured in vitro. This indicates the nature of the correlations which exist between the male and female gametophyte of Juniperus communis.     

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel imino-acid carrier in the enterocyte basolateral membrane

Basolateral membrane vesicles prepared from rat small intestinal epithelial cells were used to study the sodium-independent transport of L-proline. The uptake of L-proline was unaffected by the presence of sodium and showed saturation kinetics (Kt = 0.5 mM and Vmax = 23.3 pmol/mg protein per s). Competition experiments indicated that other amino acids had an affinity for the carrier system with L-leucine greater than L-alanine greater than sarcosine greater than glycine greater than L-lysine greater than OH-proline greater than taurine greater than beta-alanine greater than D-alanine greater than D-proline greater than L-serine greater than phenylalanine greater than valine greater than D-serine greater than phenylalanine greater than valine greater than D-serine greater than MeAIB greater than methionine greater than threonine. This pathway does not resemble those previously described either in the brush-border membrane of intestinal epithelial cells or the plasma membrane of other cell types. The lack of effect of methionine and threonine indicate that proline is not using the L-type system, while the very low affinity for MeAIB and the Na+ independence suggest that this is a novel system for imino acids. The relatively high capacity of this system and its low Kt, which is almost identical to the proline system in the brush-border membrane, strongly suggest that this is an important pathway in the final step for proline absorption by the small intestine.     

Role of threonine residue 154 in ligand recognition of the tar chemoreceptor in Escherichia coli.

The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate, maltose, and phenol, repellent responses to Ni2+ and Co2+, and thermoresponses. To understand the role of threonine residue 154, which is located in the ligand-binding domain of Tar, we replaced the residue with serine, isoleucine, and proline by site-directed mutagenesis. The replacements caused reductions in aspartate sensing but had only a small effect on maltose sensing and almost no effect on phenol sensing, repellent sensing, and thermosensing. These results indicate that Thr-154 of Tar is rather specifically involved in aspartate sensing. The reductions in the response threshold for aspartate by the replacements with serine, isoleucine, and proline were less than 1, about 2, and more than 5 orders of magnitude, respectively. When the corresponding threonine residue in the Tsr chemoreceptor was replaced with the same amino acids, roughly similar reductions in the response threshold for serine resulted. Thus, these threonine residues seem to have a common role in detecting the aspartate and serine attractant families. A mechanism by which these chemoreceptors detect the amino acid attractants is discussed.     

Effects of amino acid additions on ammonium stressed CHO cells

Ammonium is a toxic and inhibitory byproduct of mammalian cell metabolism. At the end of a typical recombinant protein production campaign, the ammonium concentration can be as high as 10 mM, mainly due to glutamine metabolism. Intracellular pH (pH(i)) levels are sensitive to ammonium, which negatively impacts both cell growth and recombinant protein productivity. Ammonium also negatively affects the recombinant protein glycosylation profile, thus altering quality. Many strategies have been adopted to reduce ammonium accumulation, with limited results. This study investigated the addition of amino acids to the growth media for Chinese hamster ovary (CHO) cell cultures as a means of mitigating the negative effects of ammonium. Threonine, proline, and glycine additions improved CHO cell growth and recombinant protein levels. Further, the threonine, proline, and glycine additions positively impacted important metabolic parameters, including glucose consumption, lactate production, glutamine utilization, and final ammonium levels. Additionally, threonine, proline, and glycine increased the level of alpha(2,3)-linked sialic acid, galactose-beta(1,4)-N-acetylglucosamine, and alpha(2,6)-linked sialic acid residues on the recombinant tissue plasminogen activator (t-PA). Thus, threonine, proline, and glycine can be used to mitigate some of the toxic effects of ammonium on cell growth, recombinant protein productivity, and protein quality.