scholarly journals Detection and Partial Purification of a Natural Heparin Inhibitor

1977 ◽  
Author(s):  
Mingjien Chien ◽  
Robert H. Yue ◽  
Menard M. Gertler
Keyword(s):  
Small Intestine ◽  
Ammonium Sulfate ◽  
Specific Activity ◽  
Partial Purification ◽  
Naturally Occurring ◽  
Antiheparin Activity ◽  
Minor Peak ◽  
Ammonium Sulfate Precipitate ◽  
Covalently Linked ◽  
A Minor

A naturally occurring heparin inhibitor has been detected in the mucosa of the fresh hog small intestine and has been partially purified. After the homogenized mucosa was extracted with Tris buffer overnight (3°) and the resulting supernatant was fractionated with ammonium sulfate, a large quantity of antiheparin activity was detected in the ammonium sulfate precipitate. This precipitate contains antiheparin activity with a specific activity of 0. 68 unit/mg of protein. Therefore, each hog small intestine contains an amount of this inhibitor enough to inhibit approximately 20, 000 units of heparin. Further purification of this heparin inhibitor was carried out by the technique of heparin affinity chromatography (covalently linked the heparin by the cyanogen bromide procedure). Eluted by a controlled NaCl and buffered gradient at 3°, the chromatogram contains a major peak and a minor peak. Antiheparin activity was located in the minor peak and has a specific activity of 9·7 units/mg of protein. Thus, we have achieved a 14-fold purification of this heparin inhibitor. This partially purified protein inhibits heparin stoichiometrically. Further experiments to purify this heparin inhibitor are in progress. This naturally occurring heparin inhibitor probably has an important biological function in balancing the action of heparin which is an important factor in maintaining Mood fluidity.

Detection and Partial Purification of a Natural Heparin Inhibitor from Hog Small Intestine

1979 ◽  
Vol 41 (03) ◽  
pp. 567-575 ◽  
Author(s):  
Menard M Gertler ◽  
Mingjien Chien ◽  
Robert H Yue
Keyword(s):  
Small Intestine ◽  
Ammonium Sulfate ◽  
Zinc Sulfate ◽  
Specific Activity ◽  
Partial Purification ◽  
Neutralizing Activity ◽  
Ethanol Precipitation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

SummaryA natural occurring heparin inhibitor was detected and was partially purified from the mucosa of hog small intestine. The mucosa was homogenized and was extracted overnight in 0.15 M NaCl, 0.01 M imidazole, 0.001 M EDTA, pH 6.5. When the extract was made to 85% saturation in ammonium sulfate, a large quantity of heparin neutralizing activity was detected in the precipitate. Each small intestine contains approximately 35,000 units of heparin neutralizing activity. This heparin inhibitor was further purified by the procedures of zinc sulfate precipitation, ammonium sulfate fractionation, ethanol precipitation and heparin-sepharose chromatography. A 37 fold partial purification with 15% overall recovery was achieved to yield heparin inhibitor with specific activity of 50-65 units per mg of protein.

scholarly journals Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum

2021 ◽  
Author(s):  
Foruzan Fathi ◽  
Rouha Kasra-Kermanshahi ◽  
Zahra Moosavi-Nejad ◽  
Elahe Mobarak Qamsari
Keyword(s):  
Enzymatic Activity ◽  
Ammonium Sulfate ◽  
High Efficiency ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Physical Adsorption ◽  
Lactobacillus Fermentum ◽  
Partial Purification ◽  
Bacterial Strains ◽  
Chitosan Beads

Background and Objectives: Due to the widespread use of lipase enzymes in various industries, finding native lipase pro- ducing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed. Materials and Methods: Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in differ- ent media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20-90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads. Results: Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respec- tively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%. Conclusion: The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

Pengaruh Penambahan Molases Pada Kulit Pisang Kapendis Untuk Media Produksi Xilanase B.Stearothermophillus DSM 22

2019 ◽  
Vol 15 (3) ◽  
Author(s):  
Trismillah
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Banana Peel ◽  
Partial Purification ◽  
Temperature Conditions ◽  
Cavendish Banana ◽  
Working Volume ◽  
Initial Ph ◽  
Xylanase Enzyme

Cavendish banana peel can be used as a substitute for the expensive xylan, while molasses than as a source of carbon as well as nitrogen, minerals and nutrients needed for the growth of microbes that can produce the enzyme. Xylanase produced from Bacillus stearothermopillus DSM 22, using media cavendish banana peels with the addition of molasses 1%, 2%, and 3%. Fermentation is done in a shaker incubator at 550C temperature conditions, initial pH 8, and 250 rpm agitation. The result showed the highest enzyme activity of 4,14 ± 0,16 U/mL min., on the addition 2% molasses after 24 hours. Further fermentation carried out in the fermenter working volume of 3.5 liters, with the condition of temperature 550C, pH 8, aeration 1 vvm, agitation 250 rpm, the highest spesific enzyme of activity of 51,62 ± 0,16 U/mg after 24 hours. Partial purification of xylanase enzyme fermentation is done with the results of microfiltration, ultrafiltration, ammonium sulfate (0-80%) and dialysis. There is an increase in the purity of the enzyme at each stage of purification, the highest purity on dialysis 3.23 times of crude enzymes.Kulit buah pisang kapendis dapat digunakan sebagai pengganti xilan yang harganya mahal, sementara molases selain sebagai sumber karbon serta nitrogen, mineral dan nutrisi dibutuhkan untuk pertumbuhan mikroba yang dapat menghasilkan enzim. Xilanase yang dihasilkan dari Bacillus stearothermopillus DSM 22, menggunakan media kulit pisang kapendis dengan penambahan molase 1%, 2%, dan 3%. Fermentasi dilakukan dalam shaker inkubator pada temperatur 550C, pH awal 8, dan agitasi 250 rpm. Hasilnya menunjukkan aktivitas enzim tertinggi 4,14 ± 0,16 U/mL min., pada penambahan 2% molases setelah 24 jam. Selanjutnya fermentasi dilakukan di dalam fermentor, volume kerja dari 3,5 liter, dengan kondisi temperatur 550C, pH 8, aeration 1 vvm, agitasi 250 rpm, aktivitas spesifik tertinggi 51,62 ± 0,16 U/mg setelah 24 jam. Pemurnian parsial fermentasi enzim xilanase dilakukan dengan hasil mikrofiltrasi, ultrafiltrasi, amonium sulfat (0-80%) dan dialisis. Ada peningkatan kemurnian enzim pada setiap tahap pemurnian, kemurnian tertinggi pada dialisis 3,23 kali dari enzim kasar.Keywords: Xylanase, B. stearothermophillus DSM 22, Cavendish banana peel, molasses, enzyme activity

scholarly journals Partial purification of presynaptic plasma membrane by immunoadsorption.

1982 ◽  
Vol 94 (1) ◽  
pp. 88-96 ◽  
Author(s):  
G P Miljanich ◽  
A R Brasier ◽  
R B Kelly
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Specific Activity ◽  
Electric Organ ◽  
Partial Purification ◽  
Vesicle Membrane ◽  
Specific Activities ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

Disaccharidase Deficiency

1985 ◽  
Vol 7 (3) ◽  
pp. 67-75
Author(s):  
Martin H. Ulshen
Keyword(s):  
Small Intestine ◽  
Minor Component ◽  
Building Blocks ◽  
Cow Milk ◽  
Milk Formula ◽  
First Year ◽  
Caloric Content ◽  
Dietary Component ◽  
A Minor ◽  
First Year Of Life

Disaccharidases are enzymes of the small intestine, and they are essential for normal carbohydrate digestion. Carbohydrates are an important dietary component, providing about half of the calories in a typical Western diet. The smallest carbohydrate units, the monosaccharides, are the building blocks for more complex sugars and starches. The monosaccharides of dietary importance include glucose, galactose, and fructose. Carbohydrates are present in an average diet, primarily in the form of dissacharides (two monosaccharides linked together) and starches (glucose polymers). The disaccharide lactose is the major carbohydrate in milk and accounts for about 40% of the caloric content of human milk as well as commercial cow milk formula. Lactose is composed of the monosaccharides glucose and galactose; sucrose is composed of glucose and fructose. During the first year of life, juices and solids are introduced into the diet in increasing amounts and, therefore, sucrose and starches provide an increasing proportion of the dietary calories. By the adult years, about 50% of dietary carbohydrate is ingested in the form of starch, and lactose is often a minor component of the diet. Among the dietary carbohydrates, only the monosaccharides can be transported intact across the luminal surface of the small intestine. The moroe complex carbohydrates must undergo digestion prior to assimilation.

Absorption of beta-casomorphins from autoperfused lamb and piglet small intestine

1990 ◽  
Vol 259 (3) ◽  
pp. G443-G452 ◽  
Author(s):  
L. C. Read ◽  
A. P. Lord ◽  
V. Brantl ◽  
G. Koch
Keyword(s):  
Small Intestine ◽  
Intestinal Lumen ◽  
Physiological Conditions ◽  
Naturally Occurring ◽  
Gut Epithelium ◽  
Measured Absorption ◽  
Beta Casein ◽  
Kinetics Of

beta-Casomorphins (beta-CMs) derived from milk beta-casein may exert various opiate activities in milk-fed infants. To assess the physiological significance of beta-CMs as a source of circulating opioids in infants, we measured absorption rates of several beta-CMs under near-physiological conditions using in situ autoperfused lamb intestine. The naturally occurring beta-CMs, beta-CM-7 and beta-CM-4-amide, were absorbed readily into blood with no transfer into lymph. Uptake peaked within several minutes of the luminal infusion of peptide but then declined sharply and stopped within a further 10-15 min. The recovery in blood, intestinal contents, and tissue at the end of the 30-min experiment was less than 1% of the infused dose. The low recovery was due to rapid proteolysis based on in vitro studies that demonstrated half-lives of less than 5 min in lamb blood, luminal contents, and lymph. The synthetic dipeptidyl peptidase IV-resistant analogue beta-[D-Ala2]CM- 4-amide was stable during incubation in blood, lymph, or luminal contents and was absorbed into blood at rates that were maximal within several minutes and remained steady for the 30-min period. We conclude that although natural beta-CMs are transferred across the lamb small intestine, rapid degradation within the intestinal lumen, gut epithelium, and blood would prevent entry into the circulation under normal conditions. Val-beta-CM-7, a putative stable precursor, had similar stability and kinetics of absorption to beta-CM-7, results that exclude Val-beta-CM-7 as a stable precursor for delivery of beta-CMs to the circulation. Essentially identical results to those in lambs were obtained in 7-day-old piglets.

RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel

2004 ◽  
Vol 287 (1) ◽  
pp. C36-C45 ◽  
Author(s):  
Takashi Murayama ◽  
Yasuo Ogawa
Keyword(s):  
Binding Sites ◽  
Skeletal Muscles ◽  
Specific Activity ◽  
Adenine Nucleotides ◽  
Minor Effect ◽  
Mammalian Skeletal Muscle ◽  
Selective Stabilization ◽  
Underlying Mechanisms ◽  
A Minor

We showed that frog α-ryanodine receptor (α-RyR) had a lower gain of Ca2+-induced Ca2+ release (CICR) activity than β-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective “stabilization” of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953–2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [3H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [3H]ryanodine binding (B) was normalized by the number of maximal binding sites (Bmax), whereby the specific activity of each isoform was expressed. This B/Bmax expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca2+, Mg2+, adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog α-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [3H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.

Partial purification of galactinol synthase from leaves of Cucurbita pepo

1982 ◽  
Vol 60 (7) ◽  
pp. 1054-1059 ◽  
Author(s):  
John A. Webb
Keyword(s):  
Cucurbita Pepo ◽  
Partial Purification ◽  
Galactinol Synthase ◽  
Inositol 1 ◽  
Mature Leaves ◽  
Galactosyl Transferase ◽  
Naturally Occurring ◽  
High Concentrations ◽  
Uridine Nucleotides

An enzyme synthesizing galactinol, UDP-D-galactose:myo-inositol-1-α-D-galactosyl transferase (galactinol synthase), has been isolated and partially purified from mature leaves of Cucurbita pepo. The enzyme showed optimal activity between pH 7.5 and 8.0 and required Mn2+ and the presence throughout isolation, storage, and assay of a sulfhydryl protectant (β-mercaptoethanol). EDTA was completely inhibitory. From a range of metal ions only Mg2+ partially replaced Mn2+, while Co2+, Zn2+, Cu2+, and Ni2+ were inhibitory. The uridine nucleotides and UDP-glucose were from 40 to 80% inhibitory and probably constitute part of the in vivo control system. High concentrations of galactose, melibiose, and xylose were partially inhibitory. The enzyme appeared highly specific for myo-inositol and showed no ability for galactosyl transfer to any other naturally occurring sugar or sugar alcohol. Some reactivity was obtained with the isomeric scyllo-inositol but the product was not identified. A range of other sugar nucleotides were unreactive.

Role of glucose in corneal metabolism

1985 ◽  
Vol 249 (5) ◽  
pp. C409-C416 ◽  
Author(s):  
R. S. Thies ◽  
L. J. Mandel
Keyword(s):  
Oxygen Consumption ◽  
Steady State ◽  
Specific Activity ◽  
Krebs Cycle ◽  
Lactate Production ◽  
Minor Component ◽  
Acid Oxidation ◽  
Endogenous Fatty Acid ◽  
Nonsteady State ◽  
A Minor

Glucose catabolism by glycolysis and the Krebs cycle was examined in the isolated rabbit cornea incubated with [6-14C]glucose. The production of [14C]lactate and 14CO2 from this substrate provided minimal values for the fluxes through these pathways since the tissue was in metabolic steady state but not isotopic steady state during the 40-min incubation. The specific activity of lactate under these conditions was one-third of that for [6-14C]glucose, and label dilution by exchange with unlabeled alanine was minimal, suggesting that glycogen degradation was primarily responsible for this dilution of label in the Embden-Meyerhof pathway. In addition, considerable label accumulation was found in glutamate and aspartate. Calculations revealed that these endogenous amino acid pools were not isotopically equilibrated after the incubation period, suggesting that they were responsible for the isotopic nonsteady state by exchange dilution through transaminase reactions with labeled intermediates. An estimate of glucose oxidation by the Krebs cycle, which was corrected for label dilution by exchange, indicated that glucose could account for most of the measured corneal oxygen consumption that was coupled to oxidative phosphorylation. A minor component of this respiration could not be accounted for by glucose or glycogen oxidation. Additional experiments suggested that endogenous fatty acid oxidation was probably also active under these conditions. Finally, reciprocal changes in plasma membrane Na+-K+-ATPase activity induced by ouabain and nystatin were found to concomitantly alter oxygen consumption rates and [14C]lactate production from [6-14C]glucose. These results demonstrated the capacity for regulating glycolysis and the Krebs cycle in response to changing energy demands in the cornea.

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