ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M-sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W₁ and W₂) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac-tions of W₁ and W₂ were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was up to 2-fold higher than that of myelin in the heavier subfractions of W₁ and W₂. The major myelin-associated glycoprotein was also increased in these subfractions as determined by periodic acid-Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W₁ and W₂ subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome-depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.     

酿酒酵母(Saccharomyces cerevisiae)基因启动子的分离和鉴定

用启动子探针型载体pFR109从酿酒酵母(Saccharomyces cerevisiae)总DNA中克隆到多个启动子片段,它们在大肠杆菌中均能启动β-半乳糖苷酶基因的表达。对其中Y8片段的进一步分析结果表明:该片段不仅来自酿酒酵母基因组,而且是以多拷贝的形式存在。当Y8片段再次转入供体酵母时,它仍能启动β-半乳糖苷酶基因表达,但经次克隆去掉Y8片段5′端部分顺序后,它就失去了启动子的功能。     

金鱼(红龙睛)次黄嘌呤-鸟嘌呤磷酸核糖转移酶的分离及特性初步研究

 从金鱼肝脏分离并部分纯化了HGPRT。初步结果表明,该酶的分子量为78,000左右。由3个亚基组成,每个亚基的分子量约为27,000。金鱼的HGPRT很稳定,在85℃加热10分钟,至少保留80％的酶活性。该酶与PRPP的反应产物在淀粉凝胶电泳图谱上出现两条反应带。     

溴氨酸降解菌株的分离和特性

从化工厂污泥中中分离到4个对蒽醌染料中间体溴氨酸有显降解和脱色作用的菌株。经鉴定,4株菌均为假单胞菌属（Pseudomonas sp.）。脱色效果最好的N1菌株能以溴酸为唯一碳源生长,其脱色效果受温度和pH影响较大,最佳生长条件是30℃,pH7.2。     

产棕榈油酸酵母菌的分离和鉴定

1992年分离到一株高产棕榈油酸的酵母菌。该菌株产油脂量为菌体干重的32.06％,棕榈油酸占油脂总量的52.14％,经初步鉴定认为是一株酿酒酵母Saccharomyces cerevisiae。     

四棱豆根瘤菌的分离及特性

从四棱豆根瘤上分离出一株慢生型根瘤菌(Rhizolia Ps)。在显微镜下,该菌株为革兰氏阴性,有一根亚极生鞭毛,大小为0.59×1.49微米。其增代时间为15.52小时,在39℃和含1.5％NaCl培养基中均不能生长。回接时能使四棱豆幼苗根部结瘤,固氮酶活性为2.81微摩尔乙烯·克鲜瘤~(-1)·小时~(-1)。本文对该菌株的含碳化合物利用、B.T.B.反应、在肉膏蛋白胨上的生长情况、石蕊牛奶反应、淀粉和明胶水解等生理生化特征进行了试验。     

牙龈卟啉杆菌的分离培养与鉴定

牙龈卟啉杆菌（Ｐｏｒｐｈｙｒｏｍａｎｕｓｇｉｎｇｉｖａｌｉｓ．Ｐｇ）与成年人慢性牙周炎有密切关系。我们对７６例慢性成人牙周炎龈下菌斑中的Ｐｇ菌进行了分离培养鉴定,共分离出Ｐｇ５５株。从表型特征上看Ｐｇ与其它２株卟啉杆菌极为相似,经ＤＮＡ同源性测定Ｐｇ菌具有高度同源性、且Ｐｇ抗血清特异性较强,血清学手段可作为辅助鉴定该菌的简便方法。本文还对Ｐｇ菌超显微结构进行了研究。     

CARBOHYDRATE PHYSIOLOGY OF ORCHID SEEDLINGS. II. HYDROLYSIS AND EFFECTS OF OLIGOSACCHARIDES

Orchid seedlings were grown on nutrients containing cold-sterilized sugars as sources of carbon. Action by an extra-cellular invertase was implied from the hydrolysis by seedlings of sugars containing α-D-glucopyranosyl-(1 → 2)-β-D-fructofuranoside bonds. D-galactosides (α and β) were also hydrolyzed, indicating the presence of extracellular galactosidases. Hydrolysis of α-D-fructofuranosides and α-D-galactosides decreased with increasing molecular weight. Seedling damage and growth retardation were paralleled by galactose accumulation in the substrate. Rupture of tonoplast and evagination of the nuclear envelope suggests that this sugar may alter some factor(s) responsible for the maintenance of membrane permeability.     

ISOLATION AND CHARACTERIZATION OF CHROMATIN FROM NEUROSPORA CRASSA

Different preparations of chromatin isolated from mycelia of Neurospora crassa were analyzed for DNA-associated RNA and proteins. The UV absorption spectra, the ultrastructure of chromatin, and the amino acid composition of the acid-extractable proteins were studied. The protein:DNA ratios range from 1.5 to 2.8; the RNA:DNA ratios range from 0.5 to 1.24. UV absorption shows a macimum at 259 mµ and a minimum at 238–239 mµ. The E280/E260 ranges from 0.59 to 0.70. Electron microscopy reveals a fibrous structure with individual fibers of 120–150 A average diameter. Attempts were made to study the protein by polyacrylamide gel electrophoresis and amino acid analysis. The results indicate that Neurospora chromatin does not contain basic proteins comparable to calf thymus histone. The ratios of basic to acidic amino acids range from 0.93 to 1.19. On electrophoresis, no bands are seen whose positions correspond to those of histones. Staining for basic proteins with fast green or eosin Y at pH 8.2 also shows a negative reaction, suggesting the absence of histones.     

ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D

Bovine brain cathepsin D was purified 1774-fold with a 19% recovery by affinity chromatography on immobilized pepstatin. Approximately 2 mg of enzyme protein were isolated from 150 g (wet weight) of bovine brain. The enzyme eluted from gel filtration as a single peak with a molecular weight of 40,000–42,000. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the predominant band migrated with a molecular weight of 48,000: however, less distinct bands were also present in the molecular weight ranges of 31,000 and 13,000. The isolated enzyme had isoelectric points over a range of pH 5–7 with 3 major peaks occurring at pH 5.6, 6.1, and 6.6. The amino acid composition of brain cathepsin D showed substantial differences from that reported for cathepsin D isolated from bovine spleen. Amino-terminal sequence analysis revealed an Asp-Val-lle sequence by Edman degradation. With hemoglobin as the substrate the enzyme had an apparent K, of 60mM.     

一株红假单胞菌的分离及生理特性研究

光合细菌在水处理和水生态修复领域应用前景广阔,对其生理特性研究具有重要价值。从武汉东湖分离得到一株不产氧光合细菌PUF1,通过对菌落形态、细胞超微结构、特征吸收光谱以及系统发育等分析,初步确定为红假单胞菌属(Rhodopseudomonas sp.)紫色非硫光合细菌。PUF1细胞呈直的或稍弯曲的杆状,长3.05—10.06μm,直径0.32—0.68μm,具有片层膜结构。PUF1培养物呈深紫红色,主要色素为细菌叶绿素a(Bchl. a)和类胡萝卜素。初始pH 6.0—8.0,光照强度500—3000 lx,稳定期细菌生物量无显著差异,但培养液pH>8.0会显著抑制PUF1最大光量子产量(Fv/Fm)。试验进一步研究了细菌不同生长阶段粗蛋白含量、ATP酶(ATPase)活性及Fv/Fm的变化规律。结果表明, PUF1不同生长阶段其蛋白含量有所差异,以稳定期为最高(>60%),而ATPase活性随培养时间的延长而逐渐降低。此外, PUF1光合作用与其生长状态也有一定的关系,具体表现为细菌生长周期内Fv/Fm变化规律适用单峰高斯模型,且以对数期Fv/Fm为最高。研究结果可为光合细...     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN BRAIN OF DIFFERENT SPECIES

Abstract— The uronic acid-containing glycosaminoglycans present in the brains of rat, monkey, chicken, sheep and rabbit were isolated into various fractions by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The analyses of the fractions show that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG are present in the brain of all the species studied. Hyaluronic acid is the major GAG (33–41 per cent). Chondroitin-4-sulphate (19–35 per cent), and heparan sulphate (11–19 per cent), are the next prominent GAGs, in all the species except chicken. The results indicate the similarity in the pattern of GAGs in the brain of all the species.     

ISOLATION AND CHARACTERIZATION OF THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was studied after isolation of a vesicle fraction and of vesicular subfractions by means of differential and density gradient centrifugations. The different fractions were examined electron microscopically by negative and positive staining; their content in protein and phospholipid and their ability to bind Ca⁺⁺ were determined. After homogenization, differential centrifugation yielded a "sarcovesicular fraction" (SVF) which was mainly composed of numerous vesicles of different types mixed with fibrous proteins and mitochondrial fragments. This SVF contained 2% of the protein and 25% of the phospholipid of the initial tissue extract. It had a high Ca⁺⁺ binding activity that was preserved for several days by storage in the presence of oxalate. After centrifugations of the SVF on sucrose density gradients, two vesicular subfractions were obtained which were characterized by different sedimentation rates, isopycnic banding, morphology, and composition in protein and phospholipid. (a) The low-density subfraction (ρ 1.10–1.12) contained a heterogeneous population of membranous structures: thick- and thin-walled vesicles, tubular formations, triads, and plasma membranes. Its content in protein and phospholipid was very low. (b) The high-density subfraction (ρ 1.13–1.17) was a very pure subfraction composed only of thin-walled vesicles. Its content in phospholipid was high and the ratio of phospholipid-phosphorus to protein was about 20. The calcium-binding activity found in the total SVF was recovered only in this latter homogeneous subfraction. The origin of these two subfractions from the SR is discussed.     

ISOLATION AND CHARACTERIZATION OF DEOXYRIBONUCLEIC ACID FROM A STRAIN OF STAPHYLOCOCCUS AUREUS.

Blobel, Hans (University of Wisconsin, Madison). Isolation and characterization of deoxyribonucleic acid from a strain of Staphylococcus aureus. J. Bacteriol. 82:425-429. 1961.-Highly polymerized deoxyribonucleic acid was extracted from washed cells of Staphylococcus aureus with a mixture of equal parts of phenol and 2 m NaCl at pH 7.4. The aqueous phase was treated twice with phenol and the deoxyribonucleic acid precipitated with an equal volume of 2-ethoxyethanol. Residual ribonucleic acid was removed by treatment with ribonuclease and subsequent dialysis. Deoxyribonucleic acid was reprecipitated with 2-ethoxyethanol. The final product contained less than 1% protein. The deoxyribonucleic acid obtained from S. aureus strain S(44) had a (adenosine + thymine)/(guanine + cytosine) base ratio of 1.98. Intrinsic viscosity in 0.15 m NaCl + 0.015 m sodium citrate was approximately 76 dl/g. The sedimentation coefficient, S(20)w, was close to 25 S.     

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

ISOLATION AND CHARACTERIZATION OF LUMINAL MEMBRANES FROM URINARY BLADDER

A method is reported for the isolation of a highly purified fraction of urinary bladder membranes containing hexagonal plaques. The method uses zonal centrifugation as the final step of fractionation. The purified fraction was characterized by its electron microscopic morphology, by its enzymatic profile, by quantitative and qualitative analysis of lipids and by the protein pattern obtained by electrophoresis in polyacrylamide sodium dodecyl sulfate gels. The fraction contains 65% lipids and 35% proteins. The major protein component has a molecular weight of 27,000 daltons. Phospholipids are more than the 54% of the total lipid weight. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the major phospholipids with 50%, 30%, and 7% of the total lipid phosphorus, respectively. The glycolipid fraction is 10% of the total lipid weight and is formed by only two components, both sulfatides. Total cholesterol makes up 36% of the total neutral lipid fraction of which cholesterol esters constitute 6%. Glycoproteins are also found to be present in the fraction.