Dipeptidyl peptidase IV from human serum: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) in normal human serum was purified 14,400-fold with a 25% yield to homogeneity. The molecular weight of the purified enzyme was approximately 110,000 on SDS-PAGE, almost the same as that of human kidney membrane-bound DPP IV. No difference was found between the two enzymes enzymologically and immunologically, either in substrate specificity, susceptibility to inhibitors, or cross-reactivity with an anti-rat kidney DPP IV antibody, or in their ability to bind adenosine deaminase. However, the N-terminal amino acid sequence of serum DPP IV lacked the transmembrane domain of the membrane-bound enzyme and started at the 39th position, serine, from the N-terminus predicted from the cDNA nucleotide sequence. These results suggest that membrane-bound DPP IV loses its transmembrane domain upon release into the serum, and that its structure on the plasma membrane is not required for its binding to adenosine deaminase.     

Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.     

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s

A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media. It had a molecular weight of 18,100 (+/-4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18,111 Da, confirmed by sequencing. Sedimentation equilibrium and gel filtration column chromatography showed that C1s alpha(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+. The C1s alpha(159)2 dimer had a sedimentation coefficient of 3.1 S. When the C1s alpha(159)2 was mixed with Clq, there was little or no interaction. Likewise, unactivated C1r2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with C1q little or no interaction was observed. When C1s alpha(159)2 was mixed with the 6.8 S C1r2 in Ca2+, a 7.5 S complex was formed, presumably the C1s alpha(159) x C1r x C1r x C1s alpha(159) tetramer. When C1q, which migrated at 10.1 S was mixed with C1s alpha(159)2 and C1r2 in the presence of Ca2+, a C1-like complex, but containing C1s alpha(159) instead of C1s, was formed which migrated at 14.0 S. This C1-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex. In the presence of immune complex, the C1r became activated. This suggested that the presence of the 159 amino acid C1s alpha domain, which held the C1r to the C1q, was sufficient to permit activation by an immune complex, even though the catalytic domains of C1s were not present.     

Large-scale identification of proteins of Haemophilus influenzae by amino acid composition analysis

Two-dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two-dimensional gel electrophoresis, for large-scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed-phase chromatography of the derivatized residues and post-column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompldent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error-robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.     

Purification, characterization and partial amino acid sequencing of two new aspartic proteinases from fresh flowers of Cynara cardunculus L

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15 000 Da whereas the chains of cardosin B migrated as bands of 34 000 Da and 14 000 Da. The partial amino acid sequences of the two cardosin revealed that they are similar but not identical, and that they differ from the previously reported cardoon proteinases named cynarases, which were assumed to be derived from a common precursor. Although the cardosins show some degree of similarity to each other, we could detect no immunological crossreactivity between them. Both cardosins were active at low pH and were inhibited by pepstatin, with Ki values of 3 nM for cardosin A and 1 nM for cardosin B, indicating that they belong to the class of aspartic proteinases. Significant differences between the two enzymes were also found for the Kcat/km values for the hydrolysis of two chromophoric synthetic peptides. The active-site ionization constants, pKe1 and pKe2, for cardosin A are 2.5 +/- 0.2 and 5.3+/- 0.2, whereas for cardosin B they are 3.73 +/- 0.09 and 6.7 +/- 0.1. The results herein described on the structural and kinetic properties of the cardosins indicate that they are the products of distinct genes which have probably arisen by gene duplication. A scheme for the proteolytic processing of the two enzymes is also proposed.     

Hydrolysis and amino acid composition of proteins

Amino acid composition analysis is a classical protein analysis method, which finds a wide application in medical and food science research and is indispensable for protein quantification. It is a complex technique, comprising two steps, hydrolysis of the substrate and chromatographic separation and detection of the residues. A properly performed hydrolysis is a prerequisite of a successful analysis. The most significant developments of the technology in the last decade consist in the (i) reduction of the hydrolysis time by the use of microwave radiation energy; (ii) improvement in the sensitivity of the residue detection, the quantification of the sensitive residues and separation of the enantiomeric forms of the amino acids; (iii) application of amino acid analysis in the large-scale protein identification by database search; and (iv) gradual replacement of the original ion exchange residue separation by reversed-phase high-performance liquid chromatography. Amino acid analysis is currently facing an enormous competition in the determination of the identity of proteins and amino acid homologs by the essentially faster mass spectrometry techniques. The amino acid analysis technology needs further simplification and automation of the hydrolysis, chromatography and detection steps to withstand the pressure exerted by the other technologies.     

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.     

The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution

The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared. The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical. Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match. Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces. We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes. The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.     

Complete amino acid sequences and phosphorylation sites, determined by Edman degradation and mass spectrometry, of rat parotid destrin- and cofilin-like proteins

Beta-adrenergic or cholinergic stimulation of the rat parotid gland was earlier shown to induce dephosphorylation of endogenous destrin- and cofilin-like proteins, which are phosphorylated in resting cells at Ser residues probably present near the N-terminals. The primary structures and phosphorylation sites were determined here. The rat destrin-like protein had a sequence 95% identical to the cDNA-derived sequence of porcine destrin. The rat cofilin-like protein was 98% identical to that of porcine cofilin. Each protein lacked the initiator Met and began with an acetylalanine residue followed by a Ser residue. The N-terminal peptides generated with endoproteinase Asp-N were isolated; they were each phosphorylated at Ser-2. Earlier work had shown that partial cleavage of the phosphorylated destrin- and cofilin-like proteins with cyanogen bromide provides unphosphorylated 16.7- and 18.3-kDa fragments, respectively. It was here confirmed that they contained all the Ser residues other than those present in the N-terminal peptides. From these observations, it was now concluded that the destrin- and cofilin-like proteins are rat parotid destrin and cofilin (non-muscle type), respectively, and that each protein is phosphorylated exclusively at Ser-2 in resting cells and dephosphorylated at this site in response to beta-adrenergic or cholinergic stimulation.     

Mass-spectrometric identification and sequence location of the ten residues of the new amino acid (gamma-Carboxyglutamic acid) in the N-terminal region of prothrombin

The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.     

Rice embryo globulins: amino-terminal amino acid sequences, cDNA cloning and expression

A globulin fraction prepared from rice embryos contained polypeptides or polypeptide groups of 49 kDa (designated REG1), 46 kDa (designated REG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequences of REG1 and the major polypeptide in the 35-kDa group were identical, suggesting that the REG1 polypeptide undergoes partial proteolytic processing that removes a carboxy-terminal region. A cDNA clone, designated pcREG2, encoding REG2 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence of REG2 was found to be 68% identical to that of the maize GLB2 globulin. Reg2 mRNA was present at high levels during embryo development for up to 14 days after flowering (DAF). Lower levels were found 20 DAF when the maturation of embryos was almost completed, and at the dry mature stage. Reg2 mRNA almost disappeared upon imbibition of isolated dry mature embryos but it was re-induced at a low level by further treatment with ABA. The expression of Reg2 was not induced by ABA in suspension-cultured cells, unlike that of Osem, one of the late embryogenesis abundant protein (LEA) genes.     

Physico-chemical properties and partial N-terminal amino acid sequence of chicken serum albumin

Studies have been made on the molecular weight, solubility, electrophoretic mobility, isoelectric point and N-terminal fragments containing 4 amino acids of the serum albumin in two strains of hens and their hybrids. In all the animals studied, the albumin had Asp as the N-terminal amino acid. Amino acid sequence in the 4-acid fragments was also identical: NH2--Asp--Ala--His--Lys. With respect to all the physico-chemical parameters investigated (except isoelectric point), proteins of the parental strains and of their hybrids did not exhibit significant differences.     

Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes

We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judging from their physicochemical and serological properties, we classified the rice alpha-amylase isoforms into two major classes, class I (A, B, Y, and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.     

Cloning and characterization of a potassium-coupled amino acid transporter

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na+ or H+ gradients. The present work extends the Na+ and H+ dogma by including the H+ and K+ paradigm. Lepidopteran insect larvae have a high K+ and a low Na+ content, and their midgut cells lack Na+/K+ ATPase. Instead, an H+ translocating, vacuolar-type ATPase generates a voltage of approximately -240 mV across the apical plasma membrane of so-called goblet cells, which drives H+ back into the cells in exchange for K+, resulting in net K+ secretion into the lumen. The resulting inwardly directed K+ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K+-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes alpha-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K+, Na+, and to a lesser extent Li+ were accepted as cotransported ions, but K+ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl(-)-dependent, and the K+/Na+ selectivity increased with hyperpolarization of oocytes, reflecting the increased K+/Na+ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na+ and Cl(-)-coupled neurotransmitter transporter family.     

Cloning, characterization and identification of the gene encoding phosphatidylinositol 4-kinase

The vast majority of AIDS-related deaths are associated with opportunistic infections. For fungal infections, there are few effective antifungals, particularly for systemic use. The discovery that very low doses of the bleomycin family of anticancer chemical congeners compromise the integrity of fungal cell walls led to our approach to identify genes that complement-cell wall defects, and develop methods to facilitate the identification of new antifungals targeted to fungal cell walls. This report describes one of the genes cloned by complementation of the blm1-1 mutation of S. cerevisiae using a YCp50-based yeast genomic library. Characterization and identification of the gene were carried out using drug screening tests, Southern hybridization analyses, DNA sequencing and DNA sequence similarity searches in databases. The gene STT4, is essential for viability and encodes a phosphatidylinositol 4-kinase that plays an important role in the phosphatidylinositol-mediated signal transduction pathway required for cell wall integrity. Like blm1-1 mutant strains, stt4 cells arrest mostly in the G2/M phase of the cell cycle. Further studies using this approach should help us understand the role of PI4-K in maintaining fungal cell-wall integrity, identify additional genes affecting potential target structures in cell walls of opportunistic fungal pathogens in AIDS patients, and assist in drug discovery and antifungal drug design.     

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.