Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein

To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.     

Critical assessment of proteome‐wide label‐free absolute abundance estimation strategies

There is a great interest in reliable ways to obtain absolute protein abundances at a proteome‐wide scale. To this end, label‐free LC‐MS/MS quantification methods have been proposed where all identified proteins are assigned an estimated abundance. Several variants of this quantification approach have been presented, based on either the number of spectral counts per protein or MS1 peak intensities. Equipped with several datasets representing real biological environments, containing a high number of accurately quantified reference proteins, we evaluate five popular low‐cost and easily implemented quantification methods (Absolute Protein Expression, Exponentially Modified Protein Abundance Index, Intensity‐Based Absolute Quantification Index, Top3, and MeanInt). Our results demonstrate considerably improved abundance estimates upon implementing accurately quantified reference proteins; that is, using spiked in stable isotope labeled standard peptides or a standard protein mix, to generate a properly calibrated quantification model. We show that only the Top3 method is directly proportional to protein abundance over the full quantification range and is the preferred method in the absence of reference protein measurements. Additionally, we demonstrate that spectral count based quantification methods are associated with higher errors than MS1 peak intensity based methods. Furthermore, we investigate the impact of miscleaved, modified, and shared peptides as well as protein size and the number of employed reference proteins on quantification accuracy.     

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.     

emPAI-assisted strategy enhances screening and assessment of Mycobacterium tuberculosis infection serological markers

Discovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value-assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC-MS/MS to analyse MTB culture filtrate proteins (MTB-CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB-CFPs – including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R-squared value ranged from 0.64 to 0.79. The only exception was ESAT-6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI-assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.     

Isolation and identification of mannose‐binding proteins and estimation of their abundance in sera from hepatocellular carcinoma patients

The interaction of glycan‐binding proteins (GBPs) and glycans plays a significant biological role that ranges from cell–cell recognition to cell trafficking, and glycoprotein targeting. The anomalies of GBPs related to the types and/or quantities were not clearly known in cancer incidence. It is imperative to identify and annotate the GBPs related with the canceration. Here the mannose‐binding proteins (MBPs) from the clinical sera were isolated and identified by the mannose‐magnetic particle conjugates and the high‐accuracy MS analysis. Seventy‐five MBPs from normal donors’ sera and 79 MBPs from hepatocellular carcinoma patients’ sera were identified and annotated. By using the stringent criteria of exponentially modified protein abundance index (emPAI) quantification, 12 MBPs were estimated to be significantly upregulated (emPAI ratio > 4) and nine MBPs were estimated to be significantly downregulated (emPAI ratio < 0.25) in the hepatocellular carcinoma sera. Real‐time quantitative PCR, Western blotting, and protein microarrays were also used to confirm the altered MBPs expression level and the specific binding between the isolated MBPs and mannose. The sequence recognition motifs and structure preference of the isolated MBPs were characterized. The functional enrichment analysis revealed that over 57% of the isolated MBPs were binding protein and the upregulated MBPs were involved in cell death, tumor progression, and macromolecular complex remodeling.     

Highly efficient "tight fit" immobilization of alpha-chymotrypsin in mesoporous MCM-41: a novel approach using precursor immobilization and activation

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.     

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

Molecular characterization of the waxy locus in sorghum

A comparison of approximately 4.5 kb of nucleotide sequence from the waxy locus (the granule-bound starch synthase I [GBSS I] locus) from a waxy line, BTxARG1, and a non-waxy line, QL39, revealed an extremely high level of sequence conservation. Among a total of 24 nucleotide differences and 9 indels, only 2 nucleotide changes resulted in altered amino acid residues. Protein folding prediction software suggested that one of the amino acid changes (Glu to His) may result in an altered protein structure, which may explain the apparently inactive GBSS I present in BTxARG1. This SNP was not found in the second waxy line, RTx2907, which does not produce GBSS I, and no other SNPs or indels were found in the approximately 4 kb of sequence obtained from RTx2907. Using one indel, the waxy locus was mapped to sorghum chromosome SBI-10, which is syntenous to maize chromosome 9; the waxy locus has been mapped to this maize chromosome. The distribution of indels in a diverse set of sorghum germplasm suggested that there are two broad types of non-waxy GBSS I alleles, each type comprising several alleles, and that the two waxy alleles in BTxARG1 and RTx2907 have evolved from one of the non-waxy allele types. The Glu/His polymorphism was found only in BTxARG1 and derived lines and has potential as a perfect marker for the BTxARG1 source of the waxy allele at the GBSS I locus. The indels correctly predicted the non-waxy phenotype in approximately 65% of diverse sorghum germplasm. The indels co-segregated perfectly with phenotype in two sorghum populations derived from crosses between a waxy and a non-waxy sorghum line, correctly identifying heterozygous lines. Thus, these indel markers or sequence-based SNP markers can be used to follow waxy alleles in sorghum breeding programs in selected pedigrees.     

低血压灌注对无心跳大鼠肺支气管肺泡灌洗液中不同白细胞及HMGB1 的影响

目的:研究低血压灌注对无心跳大鼠肺支气管肺泡灌洗液中不同白细胞及HMGB1的影响。方法:将32只180-200g SD雄性大鼠随机分成对照组[sham]、缺血30min组[30I]、缺血60 min组[60I]和缺血90 min组[90I](n=8),4组大鼠分别接受麻醉后持续机械通气,测定并统计各组支气管肺泡灌洗液中总白细胞计数及不同白细胞计数,免疫印迹法检测支气管肺泡灌洗液中HMGB1蛋白的表达。结果:总白细胞、巨噬细胞、淋巴细胞计数及HMGB1蛋白表达均随着低血压灌注时间的延长而逐渐增加,尤其在[60I]和[90I]两组,灌洗液中巨噬细胞和淋巴细胞计数有显著增高,并且巨噬细胞和淋巴细胞与HMGB1蛋白表达呈正相关。结论:本研究证实HMGB1蛋白表达和巨噬细胞,淋巴细胞计数在低血压灌注缺血期内升高,提示HMGB1蛋白可能具有驱动炎症反应的作用。     

Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides

Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.     

Lys_(10)-PAI-1融合蛋白在大肠杆菌中的表达、纯化和凝胶阻滞实验

用PCR方法构建 10个赖氨酸与纤溶酶原激活剂抑制物 1的融合基因Lys10 PAI 1,并克隆于pET2 8a(+ )和pET32a(+ )原核表达载体 .将重组表达质粒pET32 PAI和pET2 8 PAI转化大肠杆菌BL2 1(DE3) .IPTG诱导后可获得分子量分别为 6 3kD和 4 3kD的目的蛋白 ,表达蛋白占菌体蛋白2 0 %以上 ,大多数重组蛋白以不溶形式存在 .表达产物经变性、复性、超滤、透析和亲和层析等步骤 ,可以得到纯化的Trx·PAI 1和rPAI 1重组蛋白 .Western印迹结果表明 ,目的蛋白具有人PAI 1的抗原性 .凝胶阻滞实验发现 ,纯化的重组蛋白在一定条件下 ,可以与质粒结合 ,使质粒的迁移率明显改变 .研究结果表明 ,Trx·PAI 1和rPAI 1有希望成为受体介导基因转移的配体     

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Isobaric Labeling and Data Normalization without Requiring Protein Quantitation

Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI,¹ the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 μg protein as the starting material.     

Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes.

Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a cDNA expression library by complementation of an Escherichia coli trpC- PAI deficiency mutation. Genomic DNA blot hybridization analysis detected three nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence analysis of cDNA and genomic clones indicated that the PAI1 and PAI2. All three PAI polypeptides possess an N-terminal putative plastid target sequence, suggesting that these enzymes all function in plastids. The PAI1 gene is flanked by nearly identical direct repeats of approximately 350 nucleotides. Our results indicate that, in contrast to most microorganisms, the Arabidopsis PAI protein is not fused with indole-3-glycerolphosphate synthase, which catalyzes the next step in the pathway. Yeast artificial chromosome hybridization studies indicated that the PAI2 gene is tightly linked to the anthranilate synthase alpha subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to PAI1. cDNA restriction mapping and sequencing and RNA gel blot hybridization analysis indicated that all three genes are transcribed in wild-type plants. The expression of antisense PAI1 RNA significantly reduced the immunologically observable PAI protein and enzyme activity in transgenic plants. The plants expressing antisense RNA also showed two phenotypes consistent with a block early in the pathway: blue fluorescence under UV light and resistance to the anthranilate analog 6-methylanthranilate. The extreme nucleotide conservation between the unlinked PAI1 and PAI2 loci suggests that this gene family is actively evolving.     

Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.     

Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology

This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment.     

The amino acid sequence of two small ribosomal proteins from Bacillus stearothermophilus

The low-Mr proteins (tentatively called protein I and II) were purified from 2 M NaCl extracts of the Bacillus stearothermophilus ribosome. Their amino acid sequences have been determined from the peptides obtained by digestion with trypsin, chymotrypsin, and pepsin, and by cleavage with CNBr, using the micro-DABITC/PITC double-coupling method [FEBS Lett. (1978) 93, 205-214]. Protein I contains 56 residues and has an Mr of 6514. Protein II had 37 residues with an Mr of 4361. The amino acid sequence of protein I shows significant similarity to L32 from E. coli, whereas that of protein II is slightly, if at all, related to ribosomal protein L34 from E. coli.     

Heterogeneous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex

Rat brain cerebral cortex derived synaptic vesicles sedimenting on a 0.4 M sucrose solution were further fractionated according to size by column chromatography on Sephacryl-1000 and analyzed for their binding activities of antibodies directed against the vesicle-associated proteins synaptophysin, synapsin I, protein 65 and clathrin. Whereas synapsin I and particularly protein 65 and clathrin are associated with a large range of vesicle sizes, synaptophysin elutes with small vesicles only. Using monoclonal antibodies against either synaptophysin or protein 65 and polyacrylamide beads for solid matrix immunoprecipitation, significant differences could be revealed in the protein composition of the resulting vesicle populations. Whereas synapsin I is associated with both synaptophysin and protein 65 immunoprecipitated vesicle populations, synaptophysin appears to be only a minor constituent of vesicles precipitated with anti-protein 65. Vesicles precipitated with anti-synaptophysin antibodies are enriched in acetylcholine. Our results suggest that the vesicle membrane protein synaptophysin and protein 65 may not have a ubiquitous distribution among synaptic vesicles. Protein 65 containing large vesicle populations contain little synaptophysin and synaptophysin is mainly associated with synaptic vesicles of small diameter.