Multiple molecular forms of acid-stable plasmin inhibitor derived from human urinary trypsin inhibitor

It was found that cyanogen bromide (BrCN) treatment of the highly purified human urinary trypsin inhibitors (H-UTI; specific activity 1,897 U/mg protein, and L-UTI; specific activity 1,850 U/mg protein) readily produced new plasmin inhibitors with almost no loss of UTI activity. Five multiple forms of chemically cleaved inhibitors (UTIB-I, UTIB-II, UTIB-III, UTIB-IV and UTIB-V) could be isolated from BrCN-treated L-UTI by isoelectric focusing and gel filtration. These inhibitors were very acid-stable and their isoelectric points (pI) were 4.5, 4.6, 4.9, 5.1 and 6.4, respectively. The molecular weights by SDS-polyacrylamide gel electrophoresis were almost the same at about 23,000 +/- 3,000. Although these inhibitors showed both anti-plasmin and anti-trypsin activities, much higher anti-plasmin/anti-trypsin activities were observed in the cleaved inhibitors than in the parent UTI. They competitively inhibited human plasmin with Ki values of 3.0-4.1 X 10(-8) mol/l (H-D-Val-Leu-Lys-pNA substrate).     

DNA ligases from rat liver. Purification and partial characterization of two molecular forms

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.     

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Activity variation between and within two soybean trypsin inhibitor electrophoretic forms

The Kunitz soybean proteinase inhibitors (SBTI-A2) of 13 soybean pure-lines were examined for variation in antitryptic activity. Extraction of the inhibitor from individual seeds, electrophoresis on polyacrylamide gels, and excision of the inhibitor region from the gels were used to prepare inhibitor samples for assay. Protein content of eluate from the gel portions was determined by absorbance at 280 nm after applying corrections for background absorbance due to gel chemicals. Enzyme inhibition and protein content of the gel eluate were used to calculate the specific activity of the inhibitor obtained from each seed. The coefficient of variation for specific activity measurement was about 12%, and analysis of variance on data from 13 pure-lines showed significant variation in inhibitor activity from genetic and environmental sources. There appeared to be three levels of inhibition averaging 21.9, 29.2, and 39.5 specific activity units.This investigation was supported in part by funds from the Illinois Agricultural Experiment Station, National Soybean Processor's Association, and a grant (FR 07030) from the U.S. Public Health Service.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10^{? 9} M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Purification and characterization of a trypsin inhibitor from mouse seminal vesicle secretion

A Kazal-type trypsin inhibitor in mouse seminal vesicle secretion was purified to homogeneity via a series of purification steps including ammonium sulfate fractionation, affinity chromatography on a trypsin Affi-Gel 10 column, and HPLC on a reverse phase C4 column. It was shown to be a weak basic protein with an isoelectric point of 8.7 and to contain no carbohydrate. The protein had a specific activity of 184 U/mg protein in the inhibitory effect on the trypsin digestion of N-benzoyl-Pro-Phe-Arg-p-nitroanilide. Analysis of the kinetic data for the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin revealed that the protein was a competitive inhibitor with an inhibitory constant (Ki) of 0.15 nM. The molecular mass of the protein was determined to be 7 kDa by both gel chromatography and electrophoresis. Results of direct amino acid determinations indicated that this protein corresponded to the reading frame of MP12 cDNA identified from mouse prostate. We found that cleavage only at the reactive site of this protein (Arg19-Ile20) resulted in its denaturation.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10^–9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.     

Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Radioimmunological quantitation of urinary trypsin inhibitor

Using monospecific antibody to human urinary trypsin inhibitor, we developed a highly specific and sensitive radioimmunoassay (RIA) for measuring human urinary trypsin inhibitor. No cross-reactivity of the antibody with protein standard serum, which contained albumin, alpha 1-antitrypsin, haptoglobin, alpha 2-macroglobulin, transferrin, IgG and IgA, was observed. The sensitivity of the system was 10 ng of trypsin inhibitor per assay tube, and 5-10 microliters of urine was sufficient to determine the concentration of trypsin inhibitor in urine. The amounts excreted in the urine of 10 healthy men and 10 healthy women were 4.83 +/- 2.46 (mean +/- SD) and 3.86 +/- 1.35 mg/day, respectively. The correlation between estimates by RIA and those by enzymic assay was r = 0.96 (p less than 0.005). The method proposed here can be used to determine the concentration of urinary trypsin inhibitor in a small amount of biological fluids and cells.     

Isolation and partial characterization of the pancreatic secretory trypsin inhibitor in the rat

Isolation in a 55% yield of the low molecular weight pancreatic secretory trypsin inhibitor was achieved by gel filtration of an acid extract of whole inactive rat pancreas juice on Sephadex G-50 at pH 2.5 followed by desalting and ion-exchange chromatography on SP Sephadex C-50 at pH 4.5. Two distinct chromatographic fractions were obtained, labeled fraction 1 and 2. Fractions 1 and 2 showed three, respectively two, distinct closely migrating cationic bands on gel electrophoresis in barbital buffer, pH 8.6. Each fraction demonstrated one band on polyacrylamide disc electrophoresis at pH 4.6. The inhibitor is homogenous on gel filtration and on the basis of its stoichiometry with active site titrated rat anionic trypsin. Its molecular weight is approx. 6024. The amino acid composition is included. Rat pancreatic secretory trypsin inhibitor is trypsin-specific and interacts on a 1:1 molar basis with rat trypsin. It is good inhibitor of bovine trypsin but a poor inhibitor of human cationic trypsin and its binding to trypsin is reversible by acidification. Like other inhibitors of this sort, it is present in about 0.1–0.2% of the total protein content of the juice, and normally exists in its free form. A simple procedure for the production of antiserum to the inhibitor which is a poor antigen is also described.     

Purification and characterization of two forms of rat interleukin-2

Rat IL-2 produced by spleen cells in culture with concanavalin A was purified using gel filtration, hydrophobic chromatography, and ion-exchange chromatography. At least two forms of rat IL-2 were found to be separable by ion-exchange chromatography. These two forms have been designated form I and form II. Form I of rat IL-2 was purified by a factor of 1297 and found to have a pI of 6.4. Form II was purified by a factor of 669 and found to have a pI between 5.4 and 6.1. Lectin chromatography was used to demonstrate that these two forms most likely differ in the extent of glycosylation. In the presence of tunicamycin the production of form II was significantly reduced. The two forms of rat IL-2 differ in their abilities to promote a mixed-lymphocyte reaction. Their differences in glycosylation may be the reason for these differences in activity.     

Purification and characterization of a Kunitz-type trypsin inhibitor from Leaf-nosed viper venom.

A Kunitz-type trypsin inhibitor was purified from Leaf-nosed viper venom and the primary structure determined by peptide analysis. In relation to other trypsin inhibitors, the protein has an extended C-terminal segment and a distinct pattern of residue alterations at the functionally important contact sites with proteases.     

Purification and partial characterization of different forms of phosphofructokinase in man.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).     

Multiple forms of acetylcholinesterase in Allolobophora caliginosa: Purification and partial characterization

1. The authors studied certain characteristics of the acetylcholinesterase present in Allolobophora caliginosa; a good purification of the enzyme was achieved by homogenization, ultracentrifugation and then by Sephadex G-200 and DEAE-cellulose chromatographies.2. Three enzymatic forms, probably monomeric, dimeric and tetrameric were isolated. The monomeric one is quantitatively prevailing and shows a higher specific activity; it seems to be composed of of two subunits with the same molecular weight.3. The various active fractions are inhibited by eserine and hydrolyse acetylthiocholine more rapidly than butyrylthiocholine; besides, this latter does not cause substrate inhibition.4. The enzyme is classifiable as acetylcholine hydrolase (E.C. 3.1.1.7).