scholarly journals Thrombokinase of the Blood as Trypsin-Like Enzyme

1962 ◽  
Vol 45 (4) ◽  
pp. 103-113 ◽  
Author(s):  
J. H. Milstone
Keyword(s):  
Methyl Ester ◽  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Protein Band ◽  
Soybean Trypsin Inhibitor ◽  
Major Protein ◽  
Bovine Plasma ◽  
Major Protein Band

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.

Partial purification and properties of a β-glucosidase from Erwinia herbicola Y46

1975 ◽  
Vol 21 (4) ◽  
pp. 513-520 ◽  
Author(s):  
A. Garibaldi ◽  
L. N. Gibbins
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Basal Medium ◽  
Plant Diseases ◽  
Partial Purification ◽  
Major Protein ◽  
Erwinia Herbicola ◽  
Major Protein Band

A constitutive β-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant β-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight." The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-β-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against β-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of β-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells. The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0–7.5 (100% activity) and 4.5–>8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 °C, but only 25% after 30 min at 60 °C. The enzyme had a mean Km value (phloridzin) of 1.35 × 10−4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5–7.0.

scholarly journals Purification and properties of pyruvate kinase from the hepatopancreas of Carcinus maenas

1976 ◽  
Vol 153 (1) ◽  
pp. 127-134 ◽  
Author(s):  
I G Giles ◽  
P C Poat ◽  
K A Munday
Keyword(s):  
Pyruvate Kinase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Carcinus Maenas ◽  
Deae Cellulose ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Major Protein Band

Pyruvatekinase from the hepatopancreas of the common shore crab, Carcinus maenas, was purified to a specific activity of 240 units/mg of protein in the assay conditions described. 2. In one method of purification the enzymic activity could be resolved into two fractions after chromatography on DEAE-cellulose. Fructose 1, 6-diphosphate was able to effect the conversion of one form (peak 1) into the second (peak 2). 3. In the presence of a saturating concentration of fructose 1, 6-diphosphate both forms of the enzyme were kinetically similar. 4. Polyacrylamide-gel electrophoresis of the enzyme 1 day after preparation showed a single protein band. On storage at least three protein bands became visible, all of which were associated with pyruvate kinase activity. 5. Chromatography of the enzyme on Sephadex G-200 indicated a mol.wt. of 247000, but in the presence of fructose 1, 6-diphosphate the elution volume of the enzyme increased corresponding to a mol.wt. of 193000. 6 Dissociation of the enzyme in sodium dodecyl sulphate and 2-mercaptoethanol followed by polyacrylamide-gel electrophoresis produced one major protein band with a mol.wt. of 55000.

scholarly journals Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain

1987 ◽  
Vol 247 (3) ◽  
pp. 747-756 ◽  
Author(s):  
D C Bartelt ◽  
S Moroney ◽  
D J Wolff
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Bovine Brain ◽  
Major Protein ◽  
Dependent Manner ◽  
Major Protein Band

A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.

Functional specificity of jejunal brush-border pteroylpolyglutamate hydrolase in pig

1991 ◽  
Vol 260 (6) ◽  
pp. G865-G872 ◽  
Author(s):  
C. J. Chandler ◽  
D. A. Harrison ◽  
C. A. Buffington ◽  
N. A. Santiago ◽  
C. H. Halsted
Keyword(s):  
Brush Border ◽  
Protein Band ◽  
Major Protein ◽  
Functional Specificity ◽  
Major Protein Band ◽  
Regional Location ◽  
Minor Protein ◽  
A Minor ◽  
Hydrolysis Of

To determine the functional specificity of intestinal brush-border pteroylpolyglutamate hydrolase (PPH), we compared the regional location of in vivo hydrolysis of pteroyltriglutamate (PteGlu3) with the location of activity and immunoreactivity of the enzyme in the pig. After in vivo incubations, PteGlu3 hydrolytic products were recovered from intestinal segments in the jejunum but not from the ileum. Brush-border PPH activity in fractionated mucosa was 10-fold greater in the jejunum than in the ileum, whereas the activity of intracellular PPH was increased in the distal ileum. Antibodies to purified brush-border PPH identified a major protein band at 120 kDa and a minor protein band at 195 kDa in solubilized jejunal brush border. Immunohistochemistry identified the enzyme only on the brush-border surface of the jejunum, whereas an immunoblot of solubilized brush-border membranes identified brush-border PPH in the jejunum but not in the ileum. The parallel of the regional location of in vivo hydrolysis of PteGlu3 with the location of brush-border PPH activity and immunoreactivity demonstrates the functional specificity of this enzyme in folate digestion.

scholarly journals Outstanding Characteristics of Thrombokinase Isolated from Bovine Plasma

1963 ◽  
Vol 47 (2) ◽  
pp. 315-327 ◽  
Author(s):  
J. H. Milstone ◽  
N. Oulianoff ◽  
V. K. Milstone
Keyword(s):  
Methyl Ester ◽  
Continuous Flow ◽  
Deae Cellulose ◽  
Stability Curve ◽  
Bovine Plasma ◽  
Rapid Production ◽  
The Stability ◽  
Ph Range ◽  
Single Boundary ◽  
Electrophoretic Fraction

Thrombokinase has been isolated from bovine plasma by a procedure which begins with the highly purified product of a previously described method, chromatographs it on DEAE-cellulose, and then fractionates it by continuous flow electrophoresis, yielding 0.2 mg per liter of oxalated plasma. The electrophoretic fraction has shown a single boundary in the ultracentrifuge; and its esterase activity on toluenesulfonylarginine methyl ester has been about the same as that of thrombokinase previously isolated by repeated electrophoretic fractionations. Thrombokinase is a euglobulin with minimum solubility near pH 5.0. It is most stable within the pH range 7.5 to 9.5; but there is also a peak in the stability curve near pH 1.8. A few micrograms of thrombokinase per milliliter can activate prothrombin in the presence of EDTA. A few thousandths of a microgram causes rapid production of thrombin in the system: prothrombin, thrombokinase, calcium chloride, phosphatide, "accelerator." But, thrombokinase has less than 1/175 the proteolytic activity of crystallized trypsin.

scholarly journals Acid inhibition by intestinal nutrients mediated by CCK-A receptors but not plasma CCK

2001 ◽  
Vol 281 (4) ◽  
pp. G924-G930 ◽  
Author(s):  
K. C. Kent Lloyd ◽  
Jiafang Wang ◽  
Travis E. Solomon
Keyword(s):  
Acid Secretion ◽  
Gastric Acid ◽  
Trypsin Inhibitor ◽  
Acid Output ◽  
Liquid Diet ◽  
Acid Inhibition ◽  
Soybean Trypsin Inhibitor ◽  
The Individual

We examined the role of CCK-A receptors in acid inhibition by intestinal nutrients. Gastric acid and plasma CCK and gastrin levels were measured in rats with gastric and duodenal fistulas during intragastric 8% peptone and duodenal perfusion with saline, complete liquid diet (CLD; 20% carbohydrate, 6% fat, and 5% protein), and the individual components of CLD. Acid output was significantly inhibited (50–60%) by CLD, lipid, and dextrose. Plasma CCK was significantly increased by CLD (from 2.6 ± 0.3 to 4.8 ± 0.5 pM) and lipid (4.6 ± 0.5 pM). CCK levels 50-fold higher (218 ± 33 pM) were required to achieve similar acid inhibition by exogenous CCK-8 (10 nmol · kg−1 · h−1 iv). Intestinal soybean trypsin inhibitor elevated CCK (10.9 ± 2.5 pM) without inhibiting acid secretion. The CCK-A antagonist MK-329 (1 mg/kg iv) reversed acid inhibition caused by CLD, lipid, and dextrose. Peptone-stimulated gastrin (21.7 ± 1.9 pM) was significantly inhibited by CLD (14.5 ± 3.6 pM), lipid (12.3 ± 2.2 pM), and dextrose (11.9 ± 1.5 pM). Lipid and carbohydrate inhibit acid secretion by activating CCK-A receptors but not by altering plasma CCK concentrations.

Revised immunolocalization of the Na+-d-glucose cotransporter SGLT1 in rat organs with an improved antibody

2008 ◽  
Vol 295 (2) ◽  
pp. C475-C489 ◽  
Author(s):  
Daniela Balen ◽  
Marija Ljubojević ◽  
Davorka Breljak ◽  
Hrvoje Brzica ◽  
Vilim Z̆lender ◽  
...  
Keyword(s):  
Small Intestine ◽  
Protein Expression ◽  
Rat Kidney ◽  
Protein Band ◽  
Tissue Expression ◽  
Major Protein ◽  
Rat Organs ◽  
Major Protein Band ◽  
Outer Stripe ◽  
Intracellular Organelles

Previously, we characterized localization of Na+-glucose cotransporter SGLT1 ( Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Škarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913–926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of ∼75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.

scholarly journals Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5122-5129 ◽  
Author(s):  
Charles C. Chu ◽  
Rosa Catera ◽  
Katerina Hatzi ◽  
Xiao-Jie Yan ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Band ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Major Protein ◽  
Nonmuscle Myosin ◽  
Cell Extracts ◽  
Major Protein Band

Abstract Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Release of rabbit platelet histamine by serum in vitro

1962 ◽  
Vol 202 (2) ◽  
pp. 265-267 ◽  
Author(s):  
M. E. Tidball ◽  
P. A. Shore
Keyword(s):  
Methyl Ester ◽  
Trypsin Inhibitor ◽  
Calcium Ion ◽  
Histamine Content ◽  
Soybean Trypsin Inhibitor ◽  
Rabbit Platelet ◽  
Serum Factor ◽  
Rabbit Platelets

Freshly prepared defibrinated serum rapidly releases the histamine content of rabbit platelets. The presence of calcium ion is required for this action. Releasing activity of the serum factor slowly declines on aging of the serum. Release is inhibited by heparin or by tosylarginine methyl ester, but not by soybean trypsin inhibitor. Release is also promoted by thrombin, which again is inhibited by heparin. The serum factor does not appear to be preformed thrombin since thrombin is effective in the absence of calcium ion. The findings suggest that the serum factor may be blood thromboplastin which acts directly on platelets or indirectly through the generation of thrombin.

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

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