Human alpha-1-proteinase inhibitor mechanism of action: evidence for activation by limited proteolysis.

Human plasma alpha-1-proteinase inhibitor (α₁-antitrypsin) has been re-isolated from its complex with porcine trypsin. The re-isolated protein (α₁-PI^*) was found to be non-inhibitory and 8,000 lower in molecular weight than the native inhibitor. Sequence analysis of α₁-PI^* showed that an amino terminal peptide had been lost, apparently the result of cleavage at a Lys-Thr bond. These data indicate that limited proteolysis is the first step in the inhibitory mechanism.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Molecular characterization of a laminin-derived oligopeptide with implications in biomimetic applications

The molecular properties of the laminin-derived oligopeptide, H-CDPGYIGSR-NH2, have been investigated with the aid of a tandem computer simulation/experimental approach. The simulation studies placed a particular emphasis on studying the oligopeptide in aqueous media, as well as in a grafted or immobilized state. The simulations revealed the presence of a stable double hydrogen bond between arginine (R) and aspartic acid (D) residues. The mutation of the terminal arginine with lysine, another hydrophilic and positively charged amino acid, resulted in a drastic structural change, thus suggesting a major role of the terminal arginine residue in the overall oligopeptide's conformation and, hence, its bioactivity. In addition, the involvement of the aspartic acid residue in overall peptide structural stabilization also illustrates a previously undetermined role for this region (i.e. CDPG) of the oligopeptide. A subsequent in vitro experiment demonstrated a significant loss of bioactivity upon mutating the terminal residue from arginine to lysine, thereby corroborating the overall findings of the computational model.     

The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica.

The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism.     

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Isolation and properties of oxidized alpha-1-proteinase inhibitor from human rheumatoid synovial fluid

Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P₁ reactive site and the other at P₈, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed $\text{in vivo}$, presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur.     

Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.     

Biosynthesis of the cyanobacterial reserve polymer multi-L-arginyl-poly-L-aspartic acid (cyanophycin): mechanism of the cyanophycin synthetase reaction studied with synthetic primers.

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP-binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin-like polymers of 25-30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C-terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino-acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

烧伤狗浆血游离氨基酸的动态变化

烧伤狗浆血游离氨基酸的动态变化黎君友,周幼勤,赖业馥,赵有,伊少杰ＳｅｑｕｅｎｔｉａｌＣｈａｎｇｅｓｉｎＰｌａｓｍａＦｒｅｅＡｍｉｎｏＡｃｉｄＣｏｎｃｅｎｔｒａｔｉｏｎｉｎＢｕｒｎｅｄＤｏｇｓ￥Ｌｉｊｕｎｙｏｎ;Ｚｈｏｕｙｏｕｑｉｎ;Ｌａｉｙｅｆｕ;...     

Variations in the content of free and bound amino acids during dormancy and the first cell cycle in Helianthus tuberosus tuber explants

Abstract

Qualitative and quantitative analysis of free and bound amino acids and amides during dormancy and the most important phases of the first cell cycle was carried out in tubers of Helianthus tuberosus.

In the dormant tuber arginine was confirmed to be the most abundant amino acid. A high amount of asparagine was also present; on the contrary glutamine was found in very low concentrations. During the progression of dormancy, all the free amino acids and amides declined while aspartic and glutamic acid increased.

During the G₁ phase of the first cell cycle induced by 2,4-D, all the free amino acids and amides decreased with the exception of glutamic acid.

At 18, 20, 24 h of activation with 2,4-D, corresponding to the S phase and the beginning of mitosis, bound amino acids were also determined. In these phases of the cell cycle they increased reaching a maximum at 20 h; on the other hand the free amino acid and amide content, especially aspartic acid, asparagine and arginine, decreased with the exception of glutamic acid, alanine and phenylalanine.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Functional domain analysis of the Mycoplasma pneumoniae co‐chaperone TopJ

Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J‐domain co‐chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J‐domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J‐domain, central acidic and proline‐rich (APR) domain, and C‐terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J‐domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J‐domain co‐chaperone function. In contrast, TopJ derivatives having APR or C‐terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild‐type cells. Results from immunofluorescence microscopy suggest that both the APR and C‐terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.     

Structural difference between the normal PiM1 and the common PiM2 variant of human alpha 1-antitrypsin.

The common PiM2 variant of human alpha 1-antitrypsin (alpha 1-AT) which can be distinguished from the wild type PiM1 by isoelectric focusing (IEF) in a narrow pH gradient, was purified to homogeneity from plasma of a homozygous PiM2/PiM2 subject. The specific trypsin inhibitory activity and the amino acid and carbohydrate composition of the normal PiM1 and the variant PiM2 are very similar. The structural difference between the normal and the variant inhibitors was elucidated by peptide mapping of their tryptic digests. An amino acid substitution of glutamic acid in the normal inhibitor by aspartic acid in the variant inhibitor was found. The same amino acid substitution was found in PiMN, which was presumed to be identical to PiM2 based on their IEF patterns.     

Amino acid composition of triticale gluten

The study of the amino acid composition of tritikale gluten has demonstrated that in comparison with that of grain protein it contains much less aspartic acid (by 28.1%) and lysine (by 19.4%), less alanine and arginine but more tyrosine (by 39.7%), glutamic acid (by 31.4%), phenylalanine (by 23.6%) and leucine (by 14.4%) and slightly more threonine and valine. The study has shown no significant differences in the amino acid composition of grain protein and tritikale gluten of different varieties and cultivation regions.     

Fullerene‐based inhibitors of HIV‐1 protease

A series of Fmoc‐Phe(4‐aza‐C₆₀)‐OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4‐aza‐C₆₀)‐OH, is derived from the dipolar addition to C₆₀ of the Fmoc‐Nα‐protected azido amino acids derived from phenylalanine: Fmoc‐Phe(4‐aza‐C₆₀)‐Lys₃‐OH ( 1 ), Fmoc‐Phe(4‐aza‐C₆₀)‐Pro‐Hyp‐Lys‐OH ( 2 ), and Fmoc‐Phe(4‐aza‐C₆₀)‐Hyp‐Hyp‐Lys‐OH ( 3 ). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET‐based assay to evaluate the enzyme kinetics of HIV‐1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc‐Phe‐Pro‐Hyp‐Lys‐OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C₆₀‐based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and characterization of polypeptides from human plasma enhancing the growth of human normal cells in culture

The isolation in pure form and the chemical characterization is first described here of four polypeptides from human plasma which stimulate [³H] thymidine uptake into glial cells in culture and increase the number of human embryonic lung fibroblasts. The polypeptides have a molecular weight of 5000. Although they differ in charge the amino acid compositions are essentially identical and each peptide has four disulphide bridges. The amino and carboxyl terminal residues are aspartic acid and threonine respectively. The peptides are tentively designated Somatomedin B.     

The Protein Amino Acid Compositions of Amoeba proteus,A. discoides,A. dubia and Pelomyxa carolinensis*

SYNOPSIS. A comparison has been made of the protein amino acid compositions of Amoeba proteus, A. discoides, A. dubia and Pelomyxa carolinensis. The protein amino acid compositions of each of these species differed in 1–3 of the following amino acids: arginine, aspartic acid-asparagine, threonine and glycine. The possibility of using these characteristics as acceptable genetic markers is discussed.     

Mimetics of the disulfide bridge between the N- and C-terminal cysteines of the KLK3-stimulating peptide B-2

Human prostate produces kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen), which is widely used as a prostate cancer marker. Proteolytically active KLK3 has been shown to inhibit angiogenesis and its expression decreases in poorly differentiated tumors. Thus, it may be possible to control prostate cancer growth with agents that stimulate the proteolytic activity of KLK3. We have earlier developed synthetic peptides, which bind specifically to KLK3 and promote its proteolytic activity. These peptides are cyclic, all containing a disulfide bridge between the N- and C-terminal cysteines. To increase the in vivo stability of the KLK3-stimulating peptide B-2, we made differently cyclized analogues by replacing both terminal cysteines and the disulfide bridge between them. A replacement consisting of γ-amino butyric acid and aspartic acid, where the amino group from the former was linked to the main chain carboxyl group of the latter, was found to be, at high concentrations, more active than the B-2 peptide. Furthermore, as compared to the parent peptide, this analog had an improved stability in plasma and against the enzymatic degradation by KLK3. In addition, the series of analogues also provided valuable information of the structure–activity relationships of the B-2 peptide.     

Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin

A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.