Amino acid sequence of guinea pig prostate kallikrein

The primary structure of the major arginine esteropeptidase from guinea pig prostate has been deduced from automated Edman degradation of peptides generated by clostripain, cyanogen bromide, endoproteinase Lys-C, and Staphylococcus aureus V8 protease digestion of the protein. The esteropeptidase is a single polypeptide chain comprised of 239 amino acids and contains 2 apparent sites of carbohydrate attachment, Asn-78 and Asn-169. Both occur in consensus sequences for N-linked glycosylation sites. The esteropeptidase exhibits approximately 35% homology with trypsin including conservation of the catalytic residues and the aspartic acid which confers specificity toward basic amino acids. The sequence identity, however, extends to greater than 60% with the kallikrein family of serine proteases. In addition to the overall homology, the guinea pig enzyme displays a number of features characteristic of kallikreins including 10 conserved half-cystine residues, a C-terminal proline, and the "kallikrein loop". On the basis of this structural relatedness, the enzyme has been designed as guinea pig prostate kallikrein. In contrast to many of the kallikreins of other species and tissues, this enzyme does not contain any sites within the kallikrein loop sensitive to proteases that result in internal breaks in the polypeptide chain.     

Protein intake regulation in the weanling rat: Effects of additions of lysine,arginine and ammonia on the selection of gluten and energy

The effects of adding lysine, arginine and ammonia to gluten on the self-selection of protein and energy by the weanling rat simultaneously offered a choice of two diets differing only in gluten concentration (15 and 55%) were tested. Previous studies have shown that while lysine (6 g/100 g) additions to gluten decreased the amount of gluten selected by the rat from 40 to 20 g per 100 g of food eaten, selection was not related to the nutritional quality of the gluten. When graded levels of arginine (1.8, 3.6 or 7.2 g/100 g) were added to the gluten with or without lysine (0 or 6 g/100 g) the dietary protein selection was unaffected. The addition of ammonia (1.4 g/100 g as NH₄Cl) to gluten had initially the same effect as lysine (6 g/100 g) but with time protein intake returned to control levels. This effect of ammonia was unaltered by arginine additions. It is concluded that the mechanisms which lead to decreases in gluten selection caused by lysine or ammonia are not similar, and that the effects of lysine on gluten selection are not caused by an increased arginine requirement for urea cycle activity.     

Evolution of Metamorphism in Thymidylate Synthases Within the Primate Lineages

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k _cat) of mutant enzymes were 45–149% of hTS, with the lysine mutant (R163K) exhibiting the highest k _cat. The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K _d) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.     

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK_a values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK_a value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C₁-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the γ-phosphorus of ATP.     

Human–Human Hybridomas from Lung Cancer Patients Secrete Anti-DNA Antibodies

The effects of modification of the arginine/lysine ratio of dietary protein on the cholesterol kinetics were studied in male rats. Single amino acids (lysine to soybean protein and arginine to casein) were added to approximate the arginine/lysine ratio in different proteins. After acclimation to these diets for 30 days, rats were administered intravenous [¹⁴C]cholesterol and oral [³H]cholesterol. Analysis of the die-away curve of [¹⁴C]cholesterol showed an apparent independence of cholesterol kinetics to the dietary manipulations, but there was a moderate reduction of the size of the slowly exchangeable pool and of the biliary concentration of cholesterol when lysine was added to soybean protein. Addition of amino acids neither influenced cholesterol absorption nor the fecal excretion of the radioactivities from labeled cholesterol. The results indicate that manipulating the arginine/lysine ratio of dietary protein by adding single amino acids is not necessarily effective in ameliorating cholesterol metabolism in rats, although the arginine addition caused a significant reduction of serum cholesterol and triglyceride.     

Arginine and Lysine Aminopeptidase Activities in Chromaffin Granules of Bovine Adrenal Medulla: Relevance to Prohormone Processing

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH₂ and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]-enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β-mercaptoethanol (β-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β-ME/NaCl-stimulated Arg-MCA cleaving activity and β-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. K_m(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co²⁺ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni²⁺ and Zn²⁺ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.     

Purification and characterization of murine beta-nerve growth factor

Beta-nerve growth factor (β-NGF) is a trophic factor in the nervous system. We aimed to isolate and characterize this protein in view of its potential therapeutic use in neurodegenerative diseases. For purification a two-step ion-exchange procedure was followed. The characterization was performed using separation and immunological techniques, as well as a biological assay. These studies showed that the obtained protein consisted of a mixture of β-NGF molecules, intact at their NH₂-terminal extreme, and molecules which have lost the NH₂-terminal octapeptide and exhibit modifications increasing its hydrophobicity. All these molecular species were recognized immunologically and showed biological activity.     

N-Bromosuccinimide fragments of protein A24 (uH2A): an implication that ubiquitin is the precursor of conjugation in vivo

Chromatin protein A24 has a Y-branched structure in which the carboxyl terminus of ubiquitin is connected through a glycylglycine bridge to the ε-NH₂ of lysine 119 of histone 2A. In the present study, fragments of protein A24, histone 2A and ubiquitin obtained by cleavage at tryosines with N-bromosuccinimide were subjected to two-dimensional polyacrylamide gel electrophoresis and amino acid analysis. The fragmentation patterns and their compositions indicated the absence of an additional polypeptide between arginine 74 of ubiquitin and the glycyglycine bridge.     

Guinea pig MSEL-neurophysin. Sequence comparison of eight mammalian MSEL-neurophysins

The amino acid sequence of guinea pig MSEL-neurophysin has been determined using tryptic peptides derived from the performic acid-oxidized protein and staphylococcal proteinase peptides obtained from the reduced-carboxamidomethylated neurophysin. Guinea pig MSEL-neurophysin consists of a 93-residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL-neurophysin. It displays the highest number of variations among known mammalian MSEL-neurophysins. These variations are mainly found in the C-terminal region (residues 88-93). Moreover guinea pig MSEL-neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL-neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin-MSEL-neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1-9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C-terminal region of MSEL-neurophysin.     

Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer)

Two separate experiments were conducted to determine the dietary requirements of juvenile Asian sea bass Lates calcarifer Bloch for lysine and arginine. Fish (average initial weight: lysine experiment, 13.12 ± 0.12 g; arginine experiment, 2.56 ± 0.13 g) were given amino acid test diets for 12 weeks containing fish meal, zein, squid meal, and crystalline amino acids. Each set of isonitrogenous and isocaloric test diets contained graded levels of L ‐lysine or L ‐arginine. The feeding rate in the lysine experiment was at 4–2.5% of the body weight day^?1, while in the arginine experiment it was at 10–4% of the body weight day^?1. The fish (20 per tank, lysine experiment; 15 per tank, arginine experiment) were reared in 500‐L fibreglass tanks with continuous flowthrough sea water at 27 °C and salinity of 31 ppt in the lysine experiment and at 29 °C and salinity of 29 ppt in the arginine experiment. The experiments were in a completely randomized design with two replicates per treatment. Survival was high in fish given adequate lysine or arginine. Mean percentage weight gains were significantly different in fish fed varying levels of lysine or arginine. Fish fed high levels of L ‐arginine suffered high mortalities. No significant differences were obtained in the feed efficiency ratios (FER, g gain g^?1 feed) of fish fed graded lysine, although the values tended to increase as the dietary lysine level was increased up to the requirement level. In contrast, in the arginine experiment, significant differences in FER of fish among treatments were obtained; the highest FER was observed in fish fed the diet containing an optimum arginine level. On the basis of the growth response, survival, and FER, the lysine and arginine requirements of juvenile Asian sea bass were estimated to be 20.6 g kg^?1 dry diet (4.5% protein) and 18.2 g kg^?1 dry diet (3.8% protein), respectively. These data will be useful in the further refinement of practical diet formulations for the Asian sea bass.     

Crystal structures and mutational analysis of the arginine-, lysine-, histidine-binding protein ArtJ from Geobacillus stearothermophilus. Implications for interactions of ArtJ with its cognate ATP-binding cassette transporter, Art(MP)2

ArtJ is the substrate-binding component (receptor) of the ATP-binding cassette (ABC) transport system ArtJ-(MP)₂ from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine. The highest affinity is found for arginine (K_d = 0.039(±0.014) μM), while the affinities for lysine and histidine are about tenfold lower. We have determined the X-ray structures of ArtJ liganded with each of these substrates at resolutions of 1.79 Å (arginine), 1.79 Å (lysine), and 2.35 Å (histidine), respectively. As found for other solute receptors, the polypeptide chain is folded into two distinct domains (lobes) connected by a hinge. The interface between the lobes forms the substrate-binding pocket whose geometry is well preserved in all three ArtJ/amino acid complexes. Structure-derived mutational analyses indicated the crucial role of a region in the carboxy-terminal lobe of ArtJ in contacting the transport pore Art(MP)₂ and revealed the functional importance of Gln132 and Trp68. While variant Gln132Leu exhibited lower binding affinity for arginine but no binding of lysine and histidine, the variant Trp68Leu had lost binding activity for all three substrates. The results are discussed in comparison with known structures of homologous proteins from mesophilic bacteria.     

Utilization of amino acids by bacteria from the pig small intestine

This study determined the utilization of amino acids (AA) by bacteria from the lumen of the pig small intestine. Digesta samples from different segments of the small intestine were inoculated into media containing 10 mmol/L each of select AA (l-lysine, l-threonine, l-arginine, l-glutamate, l-histidine, l-leucine, l-isoleucine, l-valine, l-proline, l-methionine, l-phenylalanine or l-tryptophan) and incubated for 24 h. The previous 24-h culture served as an inoculum for a subsequent 24-h subculture during each of 30 subcultures. Results of the in vitro cultivation experiment indicated that the 24-h disappearance rates for lysine, arginine, threonine, glutamate, leucine, isoleucine, valine or histidine were 50–90% in the duodenum, jejunum or ileum groups. After 30 subcultures, the 24-h disappearance rates for lysine, threonine, arginine or glutamate remained greater than 50%. The denaturing gradient gel electrophoresis analysis showed that Streptococcus sp., Mitsuokella sp., and Megasphaera elsdenii-like bacteria were predominant in subcultures for utilizing lysine, threonine, arginine and glutamate. In contrast, Klebsiella sp. was not a major user of arginine or glutamate. Furthermore, analysis of AA composition and the incorporation of AA into polypeptides indicated that protein synthesis was a major pathway for AA metabolism in all the bacteria studied. The current work identified the possible predominant bacterial species responsible for AA metabolism in the pig small intestine. The findings provide a new framework for future studies to characterize the metabolic fate of AA in intestinal microbes and define their nutritional significance for both animals and humans.     

Classification of essential amino acids for the weanling pig

Feeding experiments with mixtures of purified amino acids show that arginine, leucine, phenylalanine, and valine are required for good growth of weanling pigs. The pig resembles the rat in its ability to synthesize part, but not all of the arginine required. It is now possible to tentatively classify the known amino acids with respect to major growth effects in weanling pigs. The amino acids which must be present in the diet for good growth are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The remaining amino acids may be tentatively classed as dispensable.     

Insulin-stimulated L-arginine transport requires SLC7A1 gene expression and is associated with human umbilical vein relaxation

Insulin causes endothelium‐derived nitric oxide (NO)‐dependent vascular relaxation, and increases L ‐arginine transport via cationic amino acid transporter 1 (hCAT‐1) and endothelial NO synthase (eNOS) expression and activity in human umbilical vein endothelium (HUVEC). We studied insulin effect on SLC7A1 gene (hCAT‐1) expression and hCAT‐transport activity role in insulin‐modulated human fetal vascular reactivity. HUVEC were used for L ‐arginine transport and L ‐[³H]citrulline formation (NOS activity) assays in absence or presence of N‐ethylmaleimide (NEM) or L ‐lysine (L ‐arginine transport inhibitors). hCAT‐1 protein abundance was estimated by Western blot, mRNA quantification by real time PCR, and SLC7A1 promoter activity by Luciferase activity (?1,606 and ?650 bp promoter fragments from ATG). Specific protein 1 (Sp1), and total or phosphorylated eNOS protein was determined by Western blot. Sp1 activity (at four sites between ?177 and ?105 bp from ATG) was assayed by chromatin immunoprecipitation (ChIP) and vascular reactivity in umbilical vein rings. Insulin increased hCATs–L ‐arginine transport, maximal transport capacity (V_max/K_m), and hCAT‐1 expression. NEM and L ‐lysine blocked L ‐arginine transport. In addition, it was trans‐stimulated (～7.8‐fold) by L ‐lysine in absence of insulin, but unaltered (～1.4‐fold) in presence of insulin. Sp1 nuclear protein abundance and binding to DNA, and SLC7A1 promoter activity was increased by insulin. Insulin increased NO synthesis and caused endothelium‐dependent vessel relaxation and reduced U46619‐induced contraction, effects blocked by NEM and L ‐lysine, and dependent on extracellular L ‐arginine. We suggest that insulin induces human umbilical vein relaxation by increasing HUVEC L ‐arginine transport via hCATs (likely hCAT‐1) most likely requiring Sp1‐activated SLC7A1 expression. J. Cell. Physiol. 226: 2916–2924, 2011. © 2011 Wiley‐Liss, Inc.     

Sequence of the regulatory region of omp T, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: implications for regulation and processing

Summary The DNA of the promoter region of ompT, including the putative start for the pro-OmpT protein (proprotein a), has been sequenced. Previous studies showed that trypsin inhibitors prevent the processing of pro-OmpT to OmpT protein which led to the prediction that the processing site would be a lysine or an arginine. The deduced amino acid sequence contains a lysine at amino acid 12 and an arginine at amino acid 17 from the N terminus. Chou-Fassman analysis would predict processing at the lysine (but not the arginine) to remove a 1389 dalton peptide, consistent with the fact that the estimated molecular masses of pro-OmpT and OmpT are 42 kd and 40 kd respectively. In addition, the predicted mRNA of the promoter region can form a stable secondary structure (-17.1 kcal) that sequesters the Shine-Dalgarno (SD) sequence as well as the initiator AUG codon. There is evidence that the perA (tpo, envZ) gene product is required for synthesis of OmpT protein (as well as several outer membrane and periplasmic proteins). The perA gene product could be activating translation of OmpT protein by disrupting the mRNA secondary structure that sequesters the SD sequence. OmpT protein synthesis is reduced at temperatures below 32°C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature.     

Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725

A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His₆-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828 ± 97 U/mg. This enzyme also racemized arginine with a specific activity of 568 ± 28 U/mg but not other amino acids. The optimal conditions for Lyr activity to l-lysine were pH 8.0–9.0 and 50 °C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5′-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5–1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine.     

Cerato-ulmin—a wilting toxin of Dutch elm disease fungus

Cerato-ulmin, a toxin produced by Ceratocystis ulmi, the causal agent of Dutch elm disease, has been characterized as a small protein (128 residues) with a MW of ca 13000. The protein has a high content of cystine, proline, leucine, serine and aspartic acid/asparagine; it is low in histidine, lysine, arginine, isoleucine, phenylalanine and tyrosine and does not contain cysteine, methionine, or tryptophan. The amino acid sequence of the N-terminal region is: H₂N-Ala-Asp-Ser-Tyr-Asp-Pro-Cys-Thr-Gly-Leu-Leu-Gln-Lys-Ser-Pro-Gln-Cys-Cys-Asp-Thr-Asp-Ile-Leu-Gly-Val-Ser-Asp-Leu-Asp-Cys-. Toxic symptoms similar to those of Dutch elm disease can be elicited by cerato-ulmin in white elm shoot cuttings (Ulmus americana L.).     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.     

Simple Branched Arginine-Based Structures can Enhance the Cellular Uptake of Peptide Cargos

In an attempt to design delivery vehicles to enable epitope-based vaccine uptake, we investigated the properties of a variety of synthetic, branched cationic structures. We found that branched compounds based on arginine or lysine were able to facilitate internalization of peptide cargo into cells to different degrees. Branched constructs containing only two arginine residues (R₂) were not only able to bind to cells more efficiently than constructs with two lysine residues (K₂) but were also internalized within vesicle like compartments in the cell. The extent of binding and uptake was enhanced when additional arginine residues were incorporated to form a tetra arginine construct (R₄). An investigation into the kinetics and dose dependence of cellular uptake of these arginine-based constructs demonstrated that binding and internalization is a rapid and efficient event. We also found uptake of the peptide epitope TYQRTRALV was enhanced when it was coupled to R₄. This approach may prove useful for introducing peptide epitopes into antigen presenting cells as self-adjuvanting structures and also for delivery of other peptides into different specialized cells.     

Expression of a VEGF-like Protein from Parapoxvirus ovis in the Yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.