Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2

The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.     

Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta

Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.     

Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis

Extracellular phospholipase A2 was purified about 1.7 X 10(5) fold to near homogeneity from human synovial fluid of rheumatoid arthritis by sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl, and reversed-phase HPLC. The final preparation showed a single band on SDS-polyacrylamide gel electrophoresis, and its molecular mass was estimated to be approximately 13,700 daltons. The purified enzyme had a pH optimum of 9.0 and required Ca2+ for maximum activity. It hydrolyzed phosphatidyl-ethanolamine more effectively than phosphatidylserine and phosphatidylcholine. These properties were similar to those of an extracellular phospholipase A2 detected in the peritoneal cavity of caseinate-treated rats.     

The NH2-terminal amino acid sequence of human proparathyroid hormone by radioisotope microanalysis.

The amino terminal amino acid sequence of human proparathyroid hormone was determined by a highly sensitive radioisotope method. Fresh human parathyroid glands were incubated with one of several ³H-labeled amino acids after which human proparathyroid hormone labeled with [³H]lysine, [³H]serine, [³H]valine, [³H]arginine, or [³H]leucine was isolated. These specimens were subjected to several cycles of Edman degradation. An increase in the amount of radioactivity liberated at any cycle was taken as evidence that the amino acid at that cycle was the one with which the sample was labeled. By this approach, we found that the amino-terminal sequence of human proparathyroid hormone is lys¹-ser²-val³-lys⁴-lys⁵-arg⁶-ser⁷-val⁸-ser⁹ … leu¹³ … leu¹⁷ … lys¹⁹ … Based on these results, we conclude that the amino-terminal region of human proparathyroid hormone consists of a hexapeptide lys-ser-val-lys-lys-arg followed by the amino acid sequence of human parathyroid hormone.     

Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid

Phospholipase A2 (PLA2) activity has now been identified in rheumatoid synovial fluids. This PLA2 is a calcium-requiring protein of MW 11,000 with a neutral pH optimum. Its activity was inhibited by high concentrations of Mg2+, and by the active site-directed histidine reagent p-bromophenacyl bromide. Ionic and nonionic detergents, or the sulfhydryl reagent dithiothreitol caused loss of enzyme activity. Synovial fluid PLA2 did not interact with sulphated mucopolysaccharides such as heparin or chondroitin sulphate. Release and sequestration of PLA2 in the joint space may contribute to the characteristic rheumatoid inflammatory changes.     

NH2-terminal amino acid distribution and amino acid composition of Streptococcus faecalis R soluble and ribosomal proteins

The NH(2)-terminal amino acid distribution of Streptococcus faecalis R soluble and ribosomal proteins isolated from cells at different stages of growth on either folate-sufficient or folate-deficient medium was determined by the dinitrophenyl method. The NH(2)-terminal residues do not follow the random distribution observed for the total amino acid composition of S. faecalis soluble and ribosomal proteins. Methionine and alanine occur most frequently; serine, threonine, aspartic and glutamic acids, and glycine are also present at the NH(2)-terminal position of S. faecalis R proteins. The absence of folic acid yields cells that are incapable of formylating methionyl-transfer ribonucelic acid tRNA(f) (Met), but does not affect either the qualitative or quantitative NH(2)-terminal distribution of total soluble or total ribosomal proteins compared to cells grown with folate. A small quantitative difference was observed in the frequency of distribution of certain amino acids at the NH(2)-termini between log and stationary phase soluble proteins. The amino acid residues found at the NH(2)-terminal position of S. faecalis proteins are qualitatively similar to those reported for several other organisms.     

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.     

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.     

The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence

The amino acid sequence of the large cyanogen bromide fragment (residues 11 to 127) derived from the NH2-terminal half of alpha-tropomyosin has been determined. This was achieved by automatic sequence analysis of the whole fragment as well as manual sequencing of fragments derived from tryptic digestion of the maleylated fragment and thermolytic, Myxobacter 495 alpha-lytic and Staphylococcus aureus protease digestion of the unmodified fragment. Methionine-containing overlap peptides have been isolated from tryptic digests of the maleylated protein as well as from S. aureus protease digests of the unmodified protein. Coupled with previously published information on the small cyanogen bromide fragments and methionine sequences of tropomyosin, these analyses have permitted the completion of the primary structure of the protein. The complete sequence differs by only 1 residue (Gln-24 instead of Glu-24) from that previously reported. Analysis of the sequence by several authors has permitted rational explanations for the stabilization of its coiled-coil structure, for the existence of its two chains in a nonstaggered arrangement, for a head-to-tail overlap of molecular ends of 8 to 9 residues, for the existence of 14 actin-binding sites on each tropomyosin molecule, and a suggestion for the site of binding of troponin-T.     

Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study

Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.     

Amino acid sequence of Trimeresurus flavoviridis phospholipase A2

The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13,764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67%) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.     

Bovine secretory component. Isolation, molecular size and shape, composition, and NH2-terminal amino acid sequence.

Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAE-cellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an (see article) of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation dquilibrium in the ultracentrifuge, using a v of 0.73 determined by ultracentrifugation in D2O and H2O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory compoennt has 2 methionine residues/mol. The NH2-terminal sequence was found to be Lys-Ser-Pro-Ile-PPHE-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that the human and dog. However, the poor immunological cross-reactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule.     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.     

Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli

A basic (pI = 10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phospholipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., & Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.     

NH2-terminal amino acid sequence of rabbit hepatopoietin A, a heparin-binding polypeptide growth factor for hepatocytes

Hepatopoietin A (HPTA) is an acidic heparin-binding polypeptide growth factor for hepatocytes with properties distinct from other known heparin-binding growth factors. HPTA is a heterodimer consisting of a heavy and a light polypeptide chain with Mr of 70,000 and 35,000 respectively. HPTA is a complete mitogen for hepatocytes in that it stimulates DNA synthesis in hepatocytes maintained in serum-free medium. Its complete purification from rabbit serum or human plasma was reported by us elsewhere (R. Zarnegar and G. Michalopoulos, 1989). In the present communication we report the N-terminal amino acid sequence of the HPTA light chain up to 24 residues (VVNGKPTRTNVGRMVSLKYRNKHI) and show that this sequence is unique and not related to any other proteins or growth factors based on computer search analysis. We have also raised antiserum against a synthetic peptide corresponding to the sequence of N-terminal amino acids residues 1 to 24, which recognizes the whole HPTA molecule. This antiserum as well as oligonucleotide probes corresponding to the N-terminal amino acid sequence of HPTA can be used as probes to identify tissue(s) of origin of this growth factor and assist in molecular cloning of its gene.