The isolation and functional identification of a protein from the human erythrocyte `ghost''

A protein, initially identified as a band on polyacrylamide-gel electrophoresis of erythrocyte ;ghosts', was isolated by selective extraction of ;ghosts' with EDTA solutions. The molecular weight of the polypeptide chain was estimated as 33000 and it represents approx. 5% of the membrane protein. The N-terminal sequence of the protein was established. Comparison with known protein sequences suggested that the protein might be the erythrocyte d-glyceraldehyde 3-phosphate dehydrogenase. This identification was confirmed by direct enzyme assay. It is suggested that this enzyme, which is strongly retained by erythrocyte ;ghosts' on haemolysis of erythrocytes, is unlikely to be an integral part of the structure of the erythrocyte membrane.     

Downstream processing of soy hull peroxidase employing reverse micellar extraction

Phase transfer studies were conducted to evaluate the solubilization of soy hull peroxidase (SHP) in reverse micelles formed in isooctane/butanol/hexanol using the cationic surfactant cetyltrimethylammonium bromide (CTAB). The effect of various parameters such as pH, ionic strength, surfactant concentration of the initial aqueous phase for forward extraction and buffer pH, type and concentration of salt, concentration of isopropyl alcohol and volume ratio for back extraction was studied to improve the efficiency of reverse micellar extraction. The active SHP was recovered after a complete cycle of forward and back extraction. A forward extraction efficiency of 100%, back extraction efficiency of 36%, overall activity recovery of 90% and purification fold of 4.72 were obtained under optimised conditions. Anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) did not yield good results under the conditions studied. The phase transfer of soy hull peroxidase was found to be controlled by electrostatic and hydrophobic interactions during forward and back extraction respectively.     

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

Glycerol permeability of human fetal and adult erythrocytes and of a model membrane

When erythrocytes from different mammalian species are compared, the hemolysis rate in 0.3 m glycerol is seen to be directly related to the percentage of lecithin in the erythrocyte phospholipid. Since this percentage is higher in erythrocytes from human adults than in those from infants, the hemolysis times in 0.3 m glycerol were compared. As expected, hemolysis was more rapid in the adult cell, which is therefore more permeable to glycerol under these conditions. The permeability to glycerol of a film of erythrocyte lipids in vitro was next examined in a model system containing the two phases water and butanol. Lipid introduced into the bulk butanol appears as a film at the interface. When equal amounts of total lipid extracted from adult and fetal erythrocytes were introduced into the butanol phase of two such chambers, the initial flux of glycerol-(14)C across the lipid boundary was greater in the cell containing lipid from adult erythrocytes than in the cell containing fetal erythrocyte lipid. This difference corresponds qualitatively to the difference in hemolysis time measured in the intact erythrocytes.     

The permeability to calcium of pigeon erythrocyte `ghosts'' studied by using the calcium-activated luminescent protein, obelin

1. Obelin, the Ca(2+)-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ;ghosts' in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca(2+) within the ;ghosts' were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ;ghosts' during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ;ghosts'. Less than 10% of the inulin or pyruvate kinase sealed within the ;ghosts' was released under any of the experimental conditions. 3. Triton X-100 (0.1-10%, v/v) made the ;ghosts' highly permeable to Ca(2+). In the presence of 1mm-Ca(2+) and Triton, 95-100% of the obelin was utilized within 10-20s. 4. A time-course of resealing ;ghosts' at 37 degrees C showed that over a period of 90min, the ;ghosts' became gradually less permeable to Ca(2+). ;Ghosts' which remained at 0 degrees C retained only a small concentration of obelin and ATP, and were highly permeable to Ca(2+). 5. Erythrocyte ;ghosts' resealed for 30min at 20 degrees C rather than 37 degrees C were more permeable to Ca(2+), as shown by the fact that 92% of the obelin in the ;ghosts' was utilized during the first 60s after the addition of 1mm-Ca(2+), as opposed to 44% for ;ghosts' resealed at 37 degrees C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ;ghosts' which were highly permeable to Ca(2+) after resealing for 60min at 37 degrees C. Of the obelin in the ;ghosts', produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca(2+) compared with 23% for ;ghosts' produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ;ghosts' to Ca(2+). Maximum effects of the ionophore (16mug/ml) were obtained by preincubating the ;ghosts' with the ionophore A23187 (16mug/ml) in the presence of a low concentration of Mg(2+) and in the absence of Ca(2+).     

Alkaline butanol extraction of bile salt and steroid sulfate esters: application to the assay of sulfotransferases

A butan-1-ol solvent-extraction procedure has been evaluated for the assay of 3'-phosphoadenosine-5'-phosphosulfate:sulfotransferase activity with various bile salt and steroid substrates. Although butanol extracted the sulfate esters of steroids and bile salts from aqueous solution at neutral pH, extraction at basic pH gave optimum recovery which was independent of protein in the sample. Greater than 99.9% of unreacted 3'-phosphoadenosine-5'-phospho[35S]sulfate remained in the aqueous phase. The data for sulfotransferase activities obtained with this solvent-extraction assay were not significantly different from those obtained with a standard thin-layer chromatography method. Solvent extraction has enabled multiple, rapid assays of several steroid and bile salt sulfotransferases during chromatographic purification of these enzymes from tissue fractions.     

Extraction of rifamycin B from native and aqueous solutions]

Optimal conditions for extraction of rifamycin B from aqueous solutions and fermentation broth filtrates at pH values within 2.0-7.0 were determined. When the antibiotic was extracted from the aqueous solutions, the highest yield was obtained at pH 2.0. When the antibiotic was extracted from the fermentation broth filtrates, it was found that chloroform was the most selective solvent with respect to rifamycin B, the chloroform selectivity being increased at pH 3.5-4.0. It was shown that rifamicin B passed from the buffer solutions with a concentration of 3-20 mg/ml to chloroform in amounts of 6-7 mg/ml and to ethylacetate and butanol in amounts of 20 mg/ml. Such conditions of chloroform and butanol (9 : 1) increased the rifamycin B contents in the extract up to 40 mg/ml.     

Rice bran lipase: extraction, activity, and stability

A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mM and pH 7, containing 0.5 mM of CaCl(2)). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability.     

Affinity chromatography of human alpha-fetoprotein on immobilized estrogens

Human alpha-fetoprotein was isolated from abortive material with the help of affinity chromatography on immobilized estrogens. After butanol extraction from the abortive material human AFP obtained the ability for affinity binding with immobilized estrogens. The addition of estrogens to the extract of isolated AFP preparation and incubation with them did not lower AFP binding with immobilized estrogens during the experiments, using affinity chromatography. A 10% buffered aqueous butanol solution was most optimal for elution. The data obtained can suggest that AFP in biological fluids is bound to estrogens, and butanol extraction deestrogenizes human AFP. The mechanism of AFP binding to estrogens in vivo is, evidently, carried out with the help of specific unknown carrier, as AFP does not bind free estrogens.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Abstract

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000?g·mol^?1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2?mL·min^?1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.     

Effect of the charge of the interface surface on the structure and activity of trypsin in reversed mycelles

The effect of the sign and value of the charge of the interface surface on the catalytic activity of trypsin in systems of reversed mycelles was investigated. n-Butanol was used for the modification of the phase interface in dispersions of reversed mycelles based on anionic sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cationic cetyltrimethylammonium bromide (CTAB). A direct correlation between changes in the state of reversed mycelles and the structure of solubilized enzyme under the action of butanol was obtained. It was shown that the enzyme activity is determined by the quantity of butanol solubilized by the reversed mycelles.     

Transport of catecholamines by resealed chromaffin-granule `ghosts''

A method is described for the preparation of resealed chromaffin-granule ;ghosts'. The lysis and rapid purification procedures provide ;ghosts' in approximately 70% yield from crude granules; the preparation contains 0.1mumol of catecholamine/mg of protein (as compared with 2.8mumol/mg in unlysed granules), of which about one third is inside the ;ghosts'. The ;ghosts' retain their ability to accumulate catecholamines, a process dependent on Mg-ATP and inhibited by reserpine, and a simple assay for this transport is described.     

Effect of the interphase surface charge on the structure and activity of trypsin in inverted micelles

The effect of the sign and value of the charge of the interphase surface on the catalytic activity of trypsin in systems of inverted mycelles was investigated. n-Butanol was used for the modification of the phase interface in dispersions of inverted mycelles based on anionic sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and cationic cetyltrimethylammonium bromide (CTAB). A direct correlation between changes in the state of inverted mycelles and the structure of solubilized enzyme under the action of butanol was obtained. It was shown that the enzyme activity is determined by the quantity of butanol solubilized by the inverted mycelles.     

The metal ion activation of the alkaline β-glycerophosphatase of rabbit small intestine

1. The preparation of gram quantities of isolated epithelial-cell ;ghosts' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs-Ringer phosphate, pH7.4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell ;ghosts' contained all of the DNA, but only 52% of the protein and 53-57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline beta-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93-116%); acid beta-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell ;ghosts'. 3. The epithelial-cell ;ghost' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15x10(4)) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3.6x10(4) and 2x10(6), and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell ;ghosts'. Two of these solutions, however, 12% dextran (mol.wt.15x10(4)) and 6% dextran (mol.wt. 2x10(6)), were too viscous to allow the complete sedimentation of the cell ;ghosts' at low relative centrifugal forces. Omission of either Krebs-Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell ;ghosts' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0.3m-sucrose-5mm-EDTA, pH7.4. The method in which cell ;ghosts' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.     

添加表面活性剂改善丁醇萃取发酵性能

研究了各种表面活性剂对丁醇萃取发酵的影响。丁醇发酵中有大量H2、CO2气体生成,生成的气泡携带发酵溶剂产物（丁醇、丙酮）进入萃取液相,促进了水相中发酵毒性产物向萃取液相的移动。研究发现,表面活性剂可以降低气-液膜的表面张力,促使大气泡破碎,从而使发酵产气以较小气泡的形式穿过萃取液相。添加表面活性剂可以强化发酵溶剂产物从水相到萃取相的移除速度,缩短发酵产物在油水两相中达到平衡的时间。有利于提高发酵生产强度。以地沟生物柴油为萃取剂,吐温-80的添加量为质量分数0．140％时,与对照相比（无表面活性剂的萃取发酵）,相同发酵时间内萃取相中丁醇体积分数提高了21．2％．总溶剂生产强唐也提高了16．5％．     

Peptidoglycan biosynthesis in Micrococcus luteus (sodonensis): transglycosidase and phosphodiesterase activities in membrane preparations.

Two enzyme activities involved in the biosynthesis of peptidoglycan in Micrococcus luteus (sodonensis), a transglycosidase and a phosphodiesterase, have been demonstrated in isolated membrane preparations. The transglycosidase activity promotes the in vitro synthesis of an uncross-bridged peptidoglycan that is completely susceptible to lysozyme. This in vitro-synthesized peptidoglycan consists of 76% "soluble" and 24% "insoluble" material. The soluble peptidoglycan is primarily a single low-molecular-weight species of approximately 20 disaccharide peptide units. "Insoluble" peptidoglycan, which likely represents newly synthesized material incorporated into an existing cell wall, was solubilized by butanol extraction, and the two were compared. The phosphodiesterase activity demonstrated in this system cleaves uridine diphosphate-N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine to yield N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-lysyl-D-alanyl-D-alanine plus uridine 5'-monophosphate plus inorganic phosphate. This phosphodiesterase activity, not detected under normal transglycosidase assay conditions, is a recycling mechanism and acts indirectly through formation and subsequent cleavage of a lipid-linked intermediate.     

Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media

Three phase partitioning (protein precipitate obtained as an interfacial layer between lower aqueous and upper t-butanol phases, formed by the addition of ammonium sulphate and t-butanol to the aqueous solution of protein) followed by lyophilization in the presence of two-component excipient resulted in 400-480x increases in transesterification activity of lyophilized powders of subtilisin Carlsberg, depending on the solvent. The three phase partitioned enzyme, 'dried' by washing with butanol, gave 3-4x higher rates (depending on the solvent used) than the enzyme preparation dried by lyophilization in the presence of two-component excipient system.