NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

A BRAIN-SPECIFIC PROTEIN FROM OCTOPUS VULGARIS, LAM

Abstract— Several major brain-specific proteins have been detected in cephalopods by electrophoretic analysis of the soluble proteins extracted from the optic lobes and other organs of octopus and by 2-dimensional fractionation of the soluble proteins from optic lobes and hepatopancreases of octopus and squid. One of the brain-specific proteins from octopus, identified as 0-1, has been purified by chromatography on DEAE-cellulose, Sephadex G-150, and DEAE-Sephadex. The protein appears to be pure on the basis of several physicochemical criteria. Amino acid analysis indicates a high content of glutamic and aspartic acids or their amides (or both) and the lack of tryptophan. A molecular weight of 17,000 has been calculated from sodium dodecyl sulphate-gel electrophoresis, gel filtration and ultracentrifugation analysis. The preparation of a specific rabbit antiserum against 0-1 has allowed its determination by agar immunodiffusion and complement fixation techniques. With the latter procedure it has been shown that the protein is absent outside the nervous system, is present in a concentration of several mg/g wet weight in octopus brain and is widely distributed within the octopus central and peripheral nervous system and in several molluscan species. It is also present in optic lobes of octopus at early stages of development.     

PROTEIN PATTERNS IN DIFFERENT LOBES AND DURING DEVELOPMENT OF OCTOPUS BRAIN

Abstract— Utilizing techniques of continuous and SDS-electrophoresis we have examined the saline-soluble and SDS-soluble (membrane-bound) proteins extracted from the main lobes of adult octopus brain and from the developing optic lobe of the same species. Several additional protein bands are present among the soluble and the membrane-bound proteins of the vertical lobe in comparison with the suboesophageal lobe. Since the former contains an essentially homogeneous population of small neurons, while the suboesophageal lobe is rich in large nerve cells, these protein bands have been attributed to the small neuronal type present in the vertical lobe.
In the course of a 10,000-fold increment in body weight, from 0.4 g to 4 kg, there is a significant increase in the concentration of several soluble proteins extracted from the optic lobe. Three of these proteins increase to a marked degree. Among the membrane-bound proteins some show a moderate increase with age while other protein components of smaller molecular weight undergo a moderate decrease. The overall tissue concentration of the membrane-bound proteins increases between 0.4 g and 50 g body weight, slightly declining in animals of larger size.     

Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.     

拮抗菌TG26的鉴定及其抗菌蛋白BI的纯化和部分特性

从丝瓜根部分离出来的桔抗菌ＴＧ２６,根据形态特征和生理生化特性,鉴定为枯草芽抱杆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）。该菌株能分泌大量的抗菌蛋白,经ＳｅｐｈａｄｅｘＧ－１５０柱和ＦＰＬＣＭｏｎｏＱ柱层析后得到单一组分的抗菌蛋白,命名为ＢＩ。ＢＩ能抑制多种植物病原菌的生长,对热稳定,对蛋白酶部分敏感。经ＳＤＳ－ＰＡＧＥ和等电聚焦电泳测定其分子量约１４．５ｋＤ,等电点为５．５８。氨基酸组成分析表明,该蛋白宫含谷氨酸、酪氨酸和脯氨酸;并测定了Ｎ末端氨基酸的部分序列。     

Isolation of eukaryotic ribosomal proteins: purification and characterization of S25 and L16.

Proteins were extracted from rat liver ribosomal subunits with ethanol and ammonium chloride. The extract from the 40S subunit contained mainly S25, but smaller amounts of a number of other proteins were found as well; the extract from the 60S subparticle had L16 in addition to P1, P2, S25, and several other proteins. S25 and L16 had not been purified before. The former was isolated from the ethanol-ammonium chloride extract by stepwise elution from carboxymethylcellulose with LiCl, chromatography on phosphocellulose, and filtration through Sephadex G-75; L16 was purified by elution from carboxymethylcellulose with LiCl (in steps). The molecular weight of the two proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; and amino acid composition was determined also.     

运动后尿液蛋白质分子量与等电点的变化特征

通过对９名男性受试者在分别完成１００－２００ｍ,４００－８００ｍ和１ ５００－３ ０００ｍ跑步间歇训练后尿蛋白分子量和等电点的测定发现：①运动时尿液高、低分子量蛋白质排泄率均较运动前明显增加,但以高分子量蛋白质排泄为主;②运动时尿液高、低分子量蛋白质排泄率均以４００－８００ｍ间歇训练时最高,１００－２００ｍ间歇训练时次之,１ ５００－３ ０００ｍ间歇训练时最低;③运动时尿液排出的蛋白质以负离子为主     

SYNTHESIS OF ACETYLCHOLINE IN DIFFERENT COMPARTMENTS OF BRAIN NERVE TERMINALS IN VIVO AS STUDIED BY THE INCORPORATION OF CHOLINE FROM PLASMA AND THE EFFECT OF PENTOBARBITAL ON THIS PROCESS

The time course of the incorporation of choline from plasma into a high and a low molecular weight fraction from mouse brain synaptosomes was studied. The fractions were obtained from lysed synaptosomes by gel filtration on Sephadex G-25. An extremely rapid incorporation of radioactivity into acetylcholine was found in both fractions and in the time interval 0.25-9 min after the intravenous administration of labelled choline, higher specific radioactivities of acetylcholine were found in the high molecular weight fraction than in the low molecular weight fraction. However, the specific radioactivity of choline in the high molecular weight fraction was much lower than that of acetylcholine. It was found that barbiturate anaesthesia caused a marked decrease in the labelling of acetylcholine in the high molecular weight fraction while the incorporation into the low molecular weight fraction was affected to a much smaller extent. Acetylcholine of the high molecular weight fraction showed properties similar to those of vesicle-bound acetylcholine. The recoveries of labelled and endogenous acetylcholine and choline from the brain homogenates were calculated in different steps of the fractionation procedure. In the fraction containing lysed synaptosomes the recovery of radioactive acetylcholine was lower than that of endogenous acetylcholine. This may indicate the presence of two types of bound acetylcholine in the synaptosomes. Different models for the intraneuronal synthesis of acetylcholine are discussed and it is proposed that a site of acetylcholine synthesis in vivo may be closely associated with some constituent of the high molecular weight fraction and directly coupled with the storage of the transmitter.     

嗜水气单胞菌S蛋白的提纯及特性分析

电镜观察表明,嗜水气单胞菌J-1株具有S层结构,在菌体外层呈晶格样规则排列。菌体经酸性甘氨酸缓冲液处理,S层从菌体上脱落,离心上清液即为粗提S蛋白。进一步经Sephadex G200凝胶层析和DEAE-纤维素离子交换层析纯化,获得的S蛋白呈单一多肽,分子量为51500。氨基酸组分分析结果表明,S蛋白含有天门冬氨酸等15种氨基酸,其中丙氨酸等疏水性氨基酸占36.8％。生物学活性显示,S蛋白对Vero细胞有轻微的细胞毒性,但没有溶血性,对鲫鱼和小鼠也无致死作用。用自制的嗜水气单胞菌J-1株S蛋白抗血清PM及PR和国外提供的嗜水气单胞菌TF7株S蛋白抗血清PF1分别作免疫转印和间接ELISA,检测来源于不同地区和不同动物种类的20株嗜水气单胞菌的S蛋白。结果表明,S蛋白的抗原性存在着菌株间的差异。另外,某些菌株不具有S层。     

中华鳖肝金属硫蛋白的分离、纯化及鉴定

金属硫蛋白metallothionein,MT)是一类低分子量、富含半胱氨酸的金属结合蛋白。MT几乎广泛分布于所有生物,包括哺乳动物、两栖动物、鱼、植物、真菌和蓝细菌,不同生物金属硫蛋白理化特性和其氨基酸序列及中心片段的比较研究,对研究MT的结构和生物功能及生物的分子进化提供重要依据。哺乳动物MT研究较多,爬行动物鳖MT的研究尚属空白,本文报道鳖肝的金属硫蛋白,中华鳖（Pelodiscus sinensis)分别经皮下注射ZnSO4,CuSO4和CdCl2溶液诱导后,取乙醇沉淀的肝脏无细胞提取液再经SephadexG-50、DEAE-SepharoseCL-6B及SephadexG-25凝胶过滤和离子交换柱层析分离,自鳖肝脏中分别获得Zn-MT、Cu-MT和Cd-MT,未经诱导的鳖肝脏中无MT,质谱和HPLC分析其分子量约为6300dalton。根据氨基酸组成分析。鳖肝脏MT含61个氨基酸残基,其中MT的典型氨基酸Cys含量占17%。Lys,Glu和Asp含量较高,而芳香族氨基酸和组氨酸含量极低,从紫外光谱特性分析,Zn-MT、Cu-MT、Cd-MT紫外吸收肩分别在220nm、270nm和250nm。表明确为鳖肝脏MT。从氨基酸残基数和分子量看,鳖肝脏MT与哺乳动物MT类似;而从氨基酸组成和结合金属离子的量看,又与低等生物蚯蚓酵母菌的MT类似。鳖MT的特性介于哺乳动物MT与低等生物MT之间,体现了鳖这种生物进化的特点。     

类产碱假单胞菌杀虫物质的分离纯化和鉴定

类产碱假单胞菌是一株昆虫病原菌,该菌对草地蝗虫、竹蝗等具有良好的致死作用,经鉴定,该菌对蝗虫具有毒杀作用的物质为其代谢分泌到胞外的一种蛋白质。类产碱假单胞菌培养物经硫酸按沉淀,SephadexG-100凝胶过滤及DEAE-SephadexA-50阴离子交换柱层析,纯化获得的杀虫蛋白只含一种亚基,分子量25100,等电点5．16,含17种氨基酸,其中谷氨酸含量最高,胱氨酸含量最低,最大吸收峰为278．3nm。     

A protein factor from Bufo marinus erythrocytes cross-bridges microtubules in vitro

A microtubule cross-bridging factor was isolated from erythrocytes of the toad, Bufo marinus. Erythrocytes were lysed and their cytoskeletons disassembled by sonication and high salt extraction. The solubilized proteins were recovered and fractionated using Sephadex G-200 column chromatography. The protein fractions from the column were analysed by SDS-PAGE and pooled into three groups: high molecular weight (HMW) proteins that eluted from the column in the void volume and had a protein composition that included HMW polypeptides; intermediate MW proteins that were shown by SDS-PAGE to contain polypeptides smaller than 120,000 D; and low MW (LMW) proteins that contained polypeptides smaller than 70,000 D. Each group was further fractionated by phosphocellulose (PC) chromatography. The flow-through was recovered, and bound proteins were then eluted by a step gradient of salt (0.2, 0.4, 0.6 and 0.8 M KCl). To assay for microtubule cross-bridging activity, column fractions were incubated with taxol-stabilized microtubules, formed from PC-purified brain tubulin (PC microtubules). Negatively stained samples were examined in the electron microscope for the reconstitution of microtubule bundles with interconnecting cross-bridges. The HMW protein fraction from the G-200 column contained the cross-bridging factor. When these proteins were further fractionated by PC chromatography only the fraction eluted by 0.2 M KCl induced the formation of microtubule bundles with cross-bridges. No other protein fraction isolated by the described method revealed cross-bridges between microtubules in vitro.     

Cadmium-binding component in Escherichia coli during accommodation to low levels of this ion.

An inducible cadmium-binding protein was isolated from Escherichia coli cells accommodated to 3 X 10(-6) M Cd2+ but not from normal or unaccommodated cells. Sephadex G-100, metal chelate affinity chromatography, and disc gel electrophoresis were used in the purification procedure. The molecular weight of the Cd2+-binding protein was estimated to be about 39,000 by Sephadex G-100 chromatography, making it different from the conventional, much smaller metallothionein.     

Plasma testosterone-binding proteins in the developing rat

Blood plasma of male rats aged 3 weeks contained two testosterone-binding proteins which differed in apparent molecular weight on Sephadex G-200 column. The binding characteristics of both proteins were similar. They had high affinity for androgens, but little or no affinity for estrogens and corticoids. Blood concentrations of both proteins began to increase markedly 15 days after birth, reached a peak at about 20 days, and then decreased. The higher molecular weight form disappeared by 5 weeks of age, whereas the lower moleculat weight form was reduced to much lower levels that remained relatively unchanged until the end of the experimental period, i.e., 63 days of age.     

Purification and Partial Characterization of Hypocalcemic Factor from Human Saliva

This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chromatography. A second gel filtration on Sephadex G-150 separates two minor salivary protein contaminants (IgA and IgG immunoglobulin) in the excluded fraction from the smaller, hypocalcemically active polypeptide.

No hypocalcemia activity could be detected or isolated in a preliminary investigation on the saliva of a dysgammaglobuli-nemic (IgA deficient) patient.

The hypocalcemia induced does not differ significantly from that observed after administration of calcitonin to mice in that: 2) minimum values are reached in 1.5–2 hours and return to normal in 5–6 hours, b) magnitude of hypocalcemia response is dose dependent. The salivary hypocalcemia factor isolated in this study has the properties of a protein, in that its activity is destroyed by the proteolytic enzyme trypsin, it yields amino acids upon acid hydrolysis and it behaves on electrophoresis, gel filtration and ion exchange chromatography as a typical protein.     

Materials with opiate receptor binding activity in bovine testis and ovine pancreas

Two groups of opiate-like materials, one with a molecular weight equal to or greater than 5000 daltons and another with a molecular weight smaller than 5000 daltons as judged by gel filtration on Sephadex G-25, were detected in bovine testes. The existence of opiate-like materials with a molecular weight smaller than 5000 daltons was demonstrated in ovine pancreas. The pancreatic fraction most strongly adsorbed on CM-cellulose possessed the highest opiate receptor binding activity. Bovine testis contained corticotropin-like material(s) which stimulated corticosterone production by isolated rat adrenal cells.     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定*

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

STUDIES ON PROTEIN METABOLISM IN HIGHER PLANTS. III. CHANGES IN THE SOLUBLE PROTEIN COMPONENTS WITH LEAF GROWTH

Water soluble protein was extracted from tobacco leaves (BrightYellow) and fractionated through chromatographic and immunochemicalprocedures. Six different UV-absorbing components, 7 antigeniccomponents, and 4 enzyme activities (phosphatase, protease,peroxidase and RNase) were detected on the Sephadex chromatogramsof leaf extracts. An UV-absorbing fraction, Fr. I-2, (immunochemicallydesignated as Pr. Imm-I) corresponded to the known "FractionI " of S. G. WILDMAN. The contents of the three antigenic components,Imm-a, Imm-b and Imm-c, having estimated molecular weights of1 to 2 10⁵, showed significant fluctuations during growthof the leaves. Peroxidase and another antigenic component (Imm-f)of smaller molecular weight showed increase with age of theleaves. Properties of the protein components thus detected wereinvestigated. (Received May 11, 1967; )     

草菇低温诱导蛋白研究初探

对低温处理的草菇Volvariella volvacea菌丝体的可溶性蛋白进行分析,发现草菇菌丝在低温协迫中有新的可溶性蛋白产生,应用电泳技术分离纯化了草菇菌丝体中的一个低温诱导蛋白,经等电聚焦分析,该蛋白质的等电点6.79,SDS-聚丙烯酰胺凝胶电泳分析该蛋白质是由分子最为70kD和48kD的两条多肽所组成。