Interaction of the mouse and bovine myelin basic proteins and two cleavage fragments with anionic detergents

The binding of deoxycholate and dodecyl sulfate to the mouse and bovine myelin basic proteins and two peptide fragments, obtained by cleavage of the bovine basic protein at its single tryptophan residue, was examined. Complete equilibrium binding isotherms for both detergents were obtained by examining their binding to each of the polypeptides immobilized on agarose. The bulk of the binding of dodecyl sulfate was found to be highly cooperative, and at saturation all four polypeptides bound far more detergent than globular, water-soluble proteins. The sum of the dodecyl sulfate bound by each of the two bovine basic protein cleavage fragments was almost twice that bound by the intact protein at saturation, suggesting that cleavage of the bovine basic protein exposes sites for additional binding of dodecyl sulfate. At pH values below pH 8.0, an additional cooperative transition was observed below the critical micelle concentration of sodium dodecyl sulfate in the binding isotherms of all four polypeptides. The midpoint of this transition corresponded to an apparent pK of approximately 5.5; however, the destruction of 90% of the histidine residues in the bovine basic protein had no effect on this transition. At pH 9.2 and moderate ionic strength (I = 0.1), the bulk of the binding of deoxycholate to the mouse and bovine basic proteins occurred at and above the critical micelle concentration of the detergent; and saturation values of deoxycholate binding to these two proteins were considerably higher than that reported for globular, water-soluble proteins. In marked contrast to the results with dodecyl sulfate, neither cleavage fragment was observed to bind deoxycholate. The results suggest that the higher ordered structure of the bovine basic protein may play an important role in the binding of anionic amphiphiles to the protein.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.     

Colvalent linkage of phospholipid to myelin basic protein: identification of phosphatidylinositol bisphosphate as the attached phospholipid

Evidence presented demonstrates a covalent attachment of a phospholipid to bovine myelin basic protein. Partial characterization of the phospholipid moiety was performed on myelin basic protein obtained from 32P-phosphorylated whole myelin that was first delipidated by two ether/ethanol (3:2 v/v) extractions, ether extraction, and acetone extraction and then purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myelin basic protein was precipitated with aqueous acetone and treated with proteases. Treatment with carboxypeptidase Y or trypsin for several hours released a lipophilic fragment, which was purified by reverse-phase high-performance liquid chromatography to yield two "lipopeptides". Such lipopeptides were obtained from both the major and minor myelin basic proteins of rat and bovine brain. Treatment with either mild base or phospholipase C removes the lipophilic character of the peptide fragment. The lipophilic fragment is a substrate for phospholipase D, but it does not comigrate on thin-layer chromatography with any 32P-labeled lipid obtained from myelin incubated with [gamma-32P]ATP. Polyphosphoinositides were shown to be released by mild acid treatment of myelin basic protein that had been extracted with organic solvent and then purified by SDS-polyacrylamide gel electrophoresis. Along with the fact that inositol monophosphate was identified in the partial acid hydrolysate of the lipopeptide, we have concluded that polyphosphoinositide (phosphatidylinositol 4-phosphate and/or phosphatidylinositol 4,5-bisphosphate) was the original phospholipid portion of the lipopeptide.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

A simple infrared spectroscopic method for the measurement of expired 13CO2.

A simple method for determination of proteins, initially solubilized in Tris buffer containing sodium dodecyl sulfate (SDS), mercaptoethanol, and sucrose, is described. This method is based on protein and Coomassie brilliant blue G-250 binding but it involves the removal of excess SDS by precipitation with 100 mm potassium phosphate buffer, pH 7.4 to 7.5, prior to protein determination. It has been established that the precipitation of excess SDS does not lead to the removal of the solubilized proteins. Therefore the method is applicable to both water-soluble and water-insoluble protein samples.     

Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.

Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.     

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.     

A lymph node neutral proteinase acting on myelin basic protein

A neutral protease present in inguinal and popliteal lymph nodes of rats with acute experimental allergic encephalomyelitis (EAE), rats injected with Freund's adjuvant, and rats that are normal has been found to hydrolyze basic protein present in purified brain and spinal cord myelin. The enzyme has been enriched by ammonium sulfate precipitation, and its properties have been studied. The protease activity toward different substrates was very specific and decreased in the following order: Protamine sulfate = polylysine (MW 183,000) > myelin basic protein > histone > polylysine (MW 2000) > polyarginine > cytochrome c. Other proteins including casein, freshly denatured hemoglobin, egg albumin, bovine serum albumin, and ribonuclease were ineffective as substrates. The pH curve showed a peak at pH7 for rat myelin, isolated beef basic protein, and histone. A possible role for this enzyme in demyelination in acute experimental allergic encephalomyelitis is suggested.     

The action of snake venom, phospholipase A and trypsin on purified myelin in vitro.

1. Purified myelin was incubated with snake venom or phospholipase A in the presence of or absence of trypsin at 37 degrees C, pH7.4, for different times. 2. Analysis of the myelin pellet obtained after centrifugation of the myelin sample incubated with snake venom or phospholipase A alone showed conversion of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine into their corresponding lyso compounds. No significant loss of myelin protein was observed in these samples. 3. A marked digestion of basic proteins and proteolipid protein was observed from the myelin pellet when trypsin was present in the incubation mixture. 4. The digestion of basic protein and particularly of proteolipid from myelin suggest that phospholipases may make protein more exposed to proteolytic enzyme for its digestion. 5. The relevance of the co-operative effect of phospholipases and proteinases as a model system of the mechanism of myelin breakdown in degenerative brain diseases is discussed.     

Detection and Characterization of Some New Basic Proteins in Chicken Postsynaptic Densities

Chicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X-100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pI greater than 9.2), 58K (pI 8.1-8.8, heterogeneous), 40K (pI 9.0), and 24K (pI 8.9). Nonequilibrium pH gradient-sodium dodecyl sulfate two-dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I-like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2+, and [gamma-32P]ATP. No PSD basic proteins bind Ca2+.     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid

A novel process is presented for the isoelectric precipitation of soy protein, using carbon dioxide as a volatile acid. By contacting a soy meal extract with pressurized carbon dioxide, the solution pH was decreased to the isoelectric region of the soy proteins. Complete precipitation of the precipitable soy proteins could be achieved for protein concentrations up to 40 g/l at pressures less than 50 bar. Isoelectric precipitation with a volatile acid enabled accurate control of the solution pH by pressure and eliminated the local pH overshoot, usual in conventional precipitation techniques. The advantage of the improved precipitation control was reflected by the morphology of the precipitate particles. Protein aggregates formed by CO₂ were perfectly spherical whereas protein precipitated by sulfuric acid had an irregular morphology. The influence of process variables to control particle size is discussed.     

人脑髓鞘碱性蛋白的分离纯化和鉴定

 本文介绍了从人脑中分离纯化髓鞘碱性蛋白的方法,人脑组织匀浆经甲醇—氯仿脱脂、酸提取、硫酸铵沉淀和羧甲基纤维素柱层析,得到了纯化的髓鞘碱性蛋白。该蛋白在SDS聚丙烯酰胺凝胶电泳中为单一带,分子量为21kD。在聚焦电泳中测得其等电点在pH10以上,氨基酸组成分析结果也与文献值接近。这为进一步研究人脑髓鞘碱性蛋白的抗原性创造了条件。     

Calcium-induced aggregation of neuroendocrine protein 7B2in vitro and its modulation by ATP

To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca²⁺ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2_156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

Isolation and Characterization of A Temperature Sensitive Protein from dog Colostrum And Milk

A temperature sensitive protein (one of several) that was soluble at 2°C but reversibly precipitated upon warming to room temperature was isolated from dog milk by precipitation with 50% saturated ammonium sulfate, ion exchange chromatography, centrifugation at 30°C and preparative isoelectric focusing in polyacrylamide gel. The molecular weight by sedimentation equilibrium was 13, 400; however, by thin layer gel filtration it appeared to be much larger. Ultracentrifu-gation studies at conditions near those at which the protein precipitated revealed no evidence of aggregation. The protein had a fairly high content of nonpolar amino acids with proline being in the highest amount. The effects of pH, ionic strength, heat and ethanol on the solubility of the protein were studied. A circular dichroism spectrum of the temperature sensitive protein indicated that the protein is quite unordered. An antiserum against the temperature sensitive protein reacted with each of the other temperature sensitive proteins from milk but not with dog serum or saliva.     

Accumulation of the four myelin basic proteins in mouse brain during development.

Myelin was isolated from the brains of mice 15, 20, 30, and 60 days after birth. The total amount of basic protein present in the isolated myelin was determined by radioimmunoassay. The 4 myelin basic proteins, with molecular weights of 21,500, 18,500, 17,000 and 14,000, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and their relative amounts were determined densitometrically. The absolute amount of each of the basic proteins was calculated from its relative amount on the gel and from the total amount of myelin basic protein in the sample as determined by radioimmunoassay. The results show that between 10 and 30 days after birth each protein accumulates at a characteristic rate so that the molar ratios among the 4 basic proteins are (in descending order according to their molecular weights) 1:5:2:10 during this period. Between 30 and 60 days after birth the 14 K and 18.5 K proteins continue to accumulate at reduced rates while the 21.5 K and 17 K proteins begin to disappear from the myelin membrane; 60 days after birth the molar ratios among the 4 basic proteins are 1:10:3.5:35. These developmental patterns of accumulation are discussed in relation to the possible role of each of the 4 myelin basic proteins in myelination.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.