Levels of alpha 1-antitrypsin and alpha 2-macroglobulin in blood serum and its antitryptic capacity

Immunochemical analysis in combination with gel filtration and isoelectric focusing made it possible to state that in blood serum of healthy people 81.3 +/- 0.5% of administered trypsin is bound with alpha 1-antitrypsin and 18.7 +/- 0.6%--with alpha 2-macroglobulin. The latter is functionally heterogeneous, only 40% of it is bound with trypsin and in the formed complex the antigenic properties of trypsin and alpha 2-macroglobulin are lost. A great number of blood serum alpha 1-antitrypsin cannot fix trypsin. The content of such alpha 1-antitrypsin rises sharply with pathology available. In the immunochemical estimation of the organism inhibitory potential relative to proteolytic enzymes not only the amount of the inhibitor but also its functional activity should be taken into account. The data of immunochemical research of the blood serum isoelectrophoregrams show that the most considerable changes under conditions of pathology occur in alpha 2-macroglobulin.     

Study of thermostable proteins in potatoes by isoelectric focusing]

With the aid of the method of isoelectric focusing eight thermostable proteins with isoelectric points of 3.6, 3.9, 4.u, 5.1, 5.8, 6.6, 7.2 and 10.0 were found in the extract from potato tubers of the Lyubimets variety. All the proteins had inhibitory activity in relation to alpha-chymotrypsin and trypsin.     

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.     

A monoclonal IgG4 (lambda) with factor V inhibitory activity.

A monoclonal IgG4 (lambda) with inhibitory activity to human coagulation factor V was isolated from the serum of a patient with a fatal hemorrhagic diathesis by using a combination of DE-52 ion exchange chromatography and isoelectric focusing techniques. Using the criteria for defining a monoclonal immunoglobulin of restricted mobility on protein electrophoresis, immunoelectrophoresis, and isoelectric focusing, as well as neutralization with class, subclass, and light chain type antisera, we are the first to demonstrate a factor V inhibitor as a monoclonal IgG4 (lambda) detectable in serum or plasma.     

Entamoeba histolytica: cytopathogenicity, including serum effects on contact-dependent and toxin-induced lysis of hamster kidney cell monolayers

Some properties of toxin, isolated from extracts of axenically cultivated Entamoeba histolytica or excreted by intact amoebae, were investigated using a toxicity assay in microplates with monolayers of baby hamster kidney cells. Preparative isoelectric focusing showed that the highest cytotoxic activity was present in a fraction of antigen containing protein bands with an isoelectric point between 4.5 and 5. Activity of the toxin was stable between pH 4 and 10. Nonimmune rabbit serum and concanavalin A, coupled to Sepharose beads, were able to bind a large amount of toxin. Cytotoxicity of antigen was inhibited by specific immune IgG and by unknown factors in nonimmune serum with a molecular weight between 50,000 and 100,000. The toxin was inactivated by trypsin, but not by trypsin inhibitor. Its activity was thiol dependent. Serum also had a marked inhibitory effect on contact lysis of BHK cells induced by intact trophozoites. A considerable reduction of both contactdependent and toxin-induced Cytopathogenicity was observed when Diamond's TP-S-1 medium was used in the assay, in which the TP broth had been autoclaved. It is suggested that Entamoeba histolytica exocytozes toxin, which acts on adjacent cells during close contact.     

Kunitz-type inhibitors in human serum. Identification and characterization

Human serum contains small amounts (approximately 0.1 mg/liter) of two protein protease inhibitors of low molecular weight (approximately 6500) and basic isoelectric point (Kunitz-type). They were purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography in the fast protein liquid chromatography system. Their chemical, immunochemical, and functional properties indicate that the purified inhibitors are highly homologous with the basic pancreatic trypsin inhibitor which is widely distributed in bovids and caprids. Their inhibitory activity toward serine proteases such as plasmin and kallikrein suggests a possible regulatory role in blood clotting and fibrinolysis.     

Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor

Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.     

ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES

A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85°C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells

Leukimia-associated inhibitory activity suppresses colony and cluster formation in vitro of cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. It was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by α-methylmannose from concanavalin A-Sepharose columns and had an apparent M_r range of 450–550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.     

Acid triacyglycerol lipase inhibitor in chicken plasma: purification and properties

1. Acid triacylglycerol lipase inhibitor was highly purified from chicken plasma by ammonium sulfate fractionation (0.6-1 saturation) followed by successive chromatographies on Hydroxyapatite, Blue-Cellulofine, Phenyl-Sepharose and Cellulofine GCL-2000 columns, and isoelectric focusing. 2. The lipase inhibitor showed its inhibitory action on triacylglycerol lipases in chicken erythrocytes ghosts and in chicken liver lysosome, but did not on pancreatic lipase, Rhizopus arrhizus lipase, or wheat germ lipase. 3. The inhibitor showed its molecular weight of 32,000 by Cellulofine GCL-2000 gel filtration. The inhibitor showed some heterogeneity on isoelectric focusing, and the main band had a pI of 5.10. 4. The lipase inhibitor did not show any inhibitory action on trypsin or chymotrypsin.     

Partial purification of proteinase inhibitors from wounded tomato plants

Proteinase inhibitors were extracted from the upper leaves of tomato plants, Lycopersicon esculentum Mill., 48 hours after wounding single lower leaves. Inhibitors were partially purified by affinity chromatography and isoelectric focusing. Significantly higher levels of trypsin and chymotrypsin inhibitory activity were recovered from wounded plants than from unwounded controls. Several inhibitor peaks were partially resolved by isoelectric focusing of affinity column eluates from both wounded and control plants. Inhibitor activity associated with each peak was greater in wounded plants than in corresponding peaks of controls. Agar double diffusion immunological assays showed that inhibitors with basic isoelectric points (pI) of 9.5, 8.9, 8.3, 8.2, and 8.0 are serologically related to inhibitor I. Certain of these inhibitors (pI = 9.5, 8.2, and 8.0) reacted strongly with both inhibitors I and II antiserum. Three acidic proteinase inhibitors (pI = 6.5, 5.9, and 4.7), which accumulated due to wounding, also were isolated. These inhibitors are novel, since they were shown to be serologically unrelated to inhibitors I and II.     

Effect of the proteinase isolated from a fungus on the kallikrein-kinin system

Carboxyl proteinase (CP) with the isoelectric point of 6.3-6.4 was isolated from a fungus at the Laboratory of Enzymology of the All-Union Research Technological Institute of Antibiotics and Enzymes and its effect on the kallikrein-kinin system and trypsin caseinolytic activity was studied. Four lots of the preparation with the activity of 1116 to 2300 milk coagulating units per 1 mg were used. The kininogenase activity of kallikrein, bradykinin and trypsin was determined with the routine biological procedures and the trypsin caseinolytic activity was determined with the Kunitz method and the diffusion method on casein-containing agar. It was shown that CP inhibited the kininogenase activity of kallikrein in the secretion of the salivary glands of man and crystalline trypsin in aqueous media and blood serum. It also inactivated the bradykinin constrictor effect on the smooth muscle tissue of the uterus horn in rats. CP had a capacity for inhibiting the caseinolytic activity of crystalline trypsin. Possible use of CP in treatment of patients with diseases accompanied by impairment of the kallikrein-kinin system (increased activity) is discussed.     

Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.     

Partial purification and chemical characterization of macrophage cytotoxicity factor (MCF, MAF) and its separation from migration inhibitory factor (MIF)

Macrophage cytotoxicity factor (MCF) was purified in 3 consecutive steps including adsorption chromatography on Matrex Gel Red A, hydrophobic chromatography on phenylalanine-Sepharose, and isoelectric focusing. MCF was characterized as a protein with a m.w. of approximately 30,000 by gel filtration on Sephadex G-100 with 2 isoelectric points at 7.4 and 8.4 in the presence of urea. The unpurified supernatant was fairly stable provided that manipulations favoring adsorption to membrane materials used for dialysis or ultrafiltration were omitted. The partially purified preparation was highly unstable. Trypsin treatment did not affect MCF activity, whereas chymotrypsin destroyed it. Treatment with glycosidases and neuraminidase or cultivation of cells in the presence of 2-deoxy-D-glucose or tunicamycin did not impair the MCF activity. MCF was separated from migration inhibitory factor (MIF) by 2 methods: first, isoelectric focusing in the presence of urea, and second by gel filtration on Ultrogel. MCF could be separated from interferon by chromatography on poly(I)-Sepharose.     

THE DIAPAUSE FACTOR FROM THE SILKWORM, BOMBYX MORI L.: PURIFICATION AND INACTIVATION EXPERIMENTS

The diapause factor, which is responsible for the induction of èmbryonic dia pause in the silkworm ( Bombyx mori L.), has been partially purified from the extract of adult heads by means of protein purification procedures, including the use of gel filtration of Sephadex, column chromatography on Dowex 1, isoelectric focusing and phenol extraction.
Two species of the diapause factor could be recognized in respect to their molecular weight. They were separated by Sephadex G-25 and their molecular weights were estimated to be about 2,000 and 5,000 from the gel filtration results. The smaller species was purified about 90-fold in specific activity, and its isoelectric point was determined by isoelectric focusing to be at about pH 4.5.
The biological activity of the partially purified principle could be abolished by incubation with several proteolytic enzymes (trypsin, α-chymotrypsin and pronase), or by treatment with amino acid-modifying reagents such as tyrosinase, N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, but was not affected by incubation with neuraminidase, cyanogen bromide or photooxidation in the presence of methylene blue.     

Purification and characterization of chymotrypsin, trypsin and elastase like proteinases from cod (Gadus morhua L.)

1. Chymotrypsin, trypsin and elastase have been purified from the pyloric caeca of cod. 2. The enzymes were separated by affinity/hydrophobic chromatography on phenyl-butyl-amine (PBA) substituted sepharose. Chymotrypsin eluted in two separate isozyme fractions whereas trypsin and elastase eluted in separate fractions consisting of two closely-related polypeptide chains as revealed by SDS-polyacrylamide electrophoresis and isoelectric focusing. 3. The cod enzymes consist of single polypeptide chains with apparent molecular weights of about 27,000 Da as shown by denaturing polyacrylamide gel electrophoresis. 4. The cod proteinases were retarded on gel filtration media. The retardation increased with increasing pressure. 5. Isoelectric focusing analysis shows that the cod enzymes have isoelectric points in the range between 5 and 7. 6. The cod proteinases are rapidly inactivated when stored at low pH's.     

Identification of a rare p.G320R alpha-1-antitrypsin variant in emphysema and lung cancer patients

The alpha-1-antitrypsin (A1AT) gene is highly polymorphic, with more than 100 genetic variants identified of which some can affect A1AT protein concentration and/or function and lead to pulmonary and/or liver disease. This study reports on the characterization of a p.G320R variant found in two patients, one with emphysema and the other with lung cancer. This variant results from a single base-pair substitution in exon 4 of the A1AT gene, and has been characterized as P by isoelectric focusing. Functional evaluation of the A1AT p.G320R variant was through comparing specific trypsin inhibitory activity in two patients with pulmonary disorders, carriers of the p.G320R variant, and 19 healthy individuals, carriers of normal A1AT M variants. Results showed that specific trypsin inhibitory activity was lower in both emphysema (2.45 mU/g) and lung cancer (2.07 mU/g) patients than in carriers of the normal variants (range 2.51-3.71 mU/g). This rare A1AT variant is associated with reduced functional activity of A1AT protein. Considering that it was found in patients with severe pulmonary disorders, this variant could be of clinical significance.     

海芋胰蛋白酶抑制剂的分离纯化及性质研究

利用亲和层析和分子筛凝胶过滤等技术,从海芋根茎中分离纯化到一种胰蛋白酶抑制剂,简称ＡＭＴＩ。经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定均显示单一条带,经ＳＤＳ－ＰＡＧＥ测定,其分子量为２２０００,经等电聚焦（ＩＥＦ）测定,其等电点为６．２。根据对胰蛋白酶的抑制比可知该抑制剂为单头抑制剂,其抑制活性在６０℃和ｐＨ５￣１１范围内保持稳定。     

Hepatoma-associated alterations of serum alpha 1-antitrypsin in hereditary hepatitis LEC rats as a new animal model of liver disease

1. The LEC rats, a novel animal model of hepatitis and liver tumor, were found to have charge variants of serum alpha 1-antitrypsin (alpha 1AT) at the stage of liver tumor as judged by isoelectric focusing. 2. Treatment of the sera with neuraminidase showed that acidic variants of alpha 1AT in LEC rats seemed to be highly sialylated forms. 3. The concentration and enzymatic activity of serum alpha 1AT in LEC rats were not affected before the onset of hepatitis and even during the development of fulminant hepatitis and hepatocarcinogenesis. This indicates that hereditary hepatitis and subsequent liver tumor in LEC rats do not appear to be associated with alpha 1AT deficiency.