The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle

A number of intracellular, membrane-bound compartments in both the endocytic and exocytic pathways of eukaryotic cells have an acidic internal pH. In endocrine cells, the mature secretory vesicle has an acidic pH; secretory vesicles isolated from exocrine cells, however, appear to have a neutral pH. Recently we have used a newly developed immunocytochemical technique to map low-pH compartments in insulin-secreting islet cells with the electron microscope and find that during the maturation of the secretory vesicle there is a progressive acidification of these vesicles that begins as soon as the trans Golgi condensing vacuoles form. Now we have used this technique to examine two exocrine cells: the pancreatic acinar cell and the parotid serous cell. In both cell types, the trans Golgi condensing vacuoles are acidic and accumulate the low-pH probe to the same extent as condensing vacuoles of insulin-secreting islet cells. Unlike insulin-secreting cells, however, maturation of the granules is accompanied by a return of luminal pH to near neutrality. Therefore, although the pH of storage granules in exocrine and endocrine cells is different, the pH of the condensing vacuoles in both cells is acidic.     

莲子贮存蛋白基因的表达与子叶叶肉细胞的变构

莲子贮存蛋白基因（ＳＰＧ）的表达受发育调控。子叶叶肉细胞变构早于ＳＰＧ的开始表达,随发育过程加剧。在贮存组织中细胞变构的发生有严格的时空顺序性。变构主要表现在细胞核体积增大,外形变不规则,染色质分布不均匀化,核膜局部消失。     

嗜铬细胞分泌的模型研究

在大鼠肾上腺嗜铬细胞上应用全细胞膜片钳和膜电容检测技术，对细胞分泌的模型进行了研究．对Ｈｅｉｎｅｍａｎｎ等提出的准备释放库Ｂ，进行了更为精确深入的分析，提出了新的论据和修改内容．证实在距细胞膜 Ｃａ２＋通道３０ｎｍ处存在立即释放库，在距Ｃａ２＋通道３００ｎｍ处存在可释放库．通过内分泌细胞分泌模型分析了Ｃａ２＋的控制作用以及囊泡释放过程的时序特征．     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

重组人甲状旁腺激素(1-34)在大肠杆菌中的表达与纯化

将化学合成的rhPTH(1-34)基因用PCR扩增后,克隆至表达载体pET-35b( ),使rhPTH(1—34)融合于纤维素结合结构域(CBDdos)的羧基端,并得到高效表达．融合蛋白经纤维素树脂亲和层析纯化后,经Factor Xa裂解释放出rhPTH(1-34),再通过纤维素树脂亲和层析、C4反向高效液相色谱纯化得到rhPTH(1-34)纯品．每升培养液可获取3mg高纯度的rhPTH(1-34)．经质谱测定,所得样品的分子量为4117．0Da,与rhPTH(1—34)理论分子量一致．     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

Isolation of a cDNA clone encoding rat insulin-like growth factor-II precursor

Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.     

'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles

Golgi-derived coated vesicles contain a set of coat proteins of relative molecular mass 160,000 (Mr 160K; alpha-COP), 110K (beta-COP), 98K (gamma-COP) and 61K (delta-COP), and several smaller subunits. We have now identified and purified a cytosolic complex containing the same four coat proteins as those of Golgi transport vesicles. We term this complex the Golgi coat promoter or 'coatomer'. The coatomer also contains polypeptides of Mr 36K, 35K and 20K. It represents about 0.2% of soluble cytosolic protein. Gel filtration of unfractionated cytosol indicates that beta-COP resides exclusively in the coatomer complex. The complex seems to be a likely candidate for the unassembled precursor of Golgi coated vesicles, and its purification should help investigations of the role of coat proteins in membrane budding, for which it is necessary to use a refined cell-free system.     

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.     

肽泡的形态特征及其成膜蛋白的特性

小麦(Triticum aestivum)贮存蛋白经过纯化获得其中35kD一种蛋白质。该蛋白质在一定浓度的有机溶剂系统中经过超声波处理或搅拌,可以制成囊泡,并能包裹分子量从0.4～150kD的不同化合物(染料及抗体)。这种肽泡具有良好的稳定性,经低温冷冻干燥后,可成鳞片状,加水后复溶仍然保持肽泡的稳定性。这种特性称为DRV(Dehydration Rehydration Vesicles)性质。该蛋白质经修饰制定肽泡能包裹水溶性化合物,如水溶性染料,或不经修饰可包裹脂溶性化合物。这种蛋白质是由16种疏水性氨基酸残基组成,其N-末端为苯丙氨酸。     

The cellular oncogene p53 can be activated by mutagenesis

P53 is a cellular phosphoprotein of short half-life (t1/2) which is present at elevated levels in cells transformed by various stimuli including viruses, chemicals and radiation. p53 forms specific stable complexes with simian virus 40 (SV40) large-T antigen and an adenovirus E1b protein of relative molecular mass (Mr) 57,000. A number of reports have associated p53 with cell proliferation, and p53 complementary DNA expression constructs immortalize primary cells in vitro and render them sensitive to transformation by an activated ras oncogene. We have examined the biological properties of a set of p53 expression constructs, and report here that cellular immortalization by a wild-type p53 cDNA gene is conditional upon the promoter/enhancer construction used, but that p53 can extend cellular lifespan by a second distinct mechanism involving rearrangements of the coding sequence which give rise to stable protein products. Cells immortalized by one of these mutants are refractory to subsequent transformation by a ras oncogene, indicating that cellular immortalization and ras cooperation are separate activities.     

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.     

Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites

Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence.     

Molecular cloning and expression of the major protein kinase C substrate of platelets

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.     

Characterization of an expressed CD3-associated Ti gamma-chain reveals C gamma domain polymorphism

The majority of human T cells express an antigen receptor consisting of a disulphide-linked heterodimer (Ti) of relative molecular mass 80,000-90,000 (Mr 80-90K) which is noncovalently associated with a set of at least three proteins of Mr 20-28K termed CD3 (Leu4, T3). Whereas both chains of Ti, an acidic alpha-chain of Mr 48-54K and a more basic beta-chain of Mr 40-44K, contain variable and constant region domains, the component peptides of CD3 are invariant. Several laboratories have more recently reported the expression of CD3 in association with a novel protein. On the surface of long-term T-cell lines and one thymocyte clone this novel structure consists of a 40K protein noncovalently linked to a 55 or 62K protein identified as the protein product of the Ti gamma-chain gene, a T-cell specific gene which like the Ti alpha- and Ti beta-chain genes undergoes rearrangement of variable (V) and joining (J) region gene segments. On the human T-cell leukaemic line PEER we have detected only a single 55K glycoprotein associated with CD3. We here demonstrate that an anti-Ti gamma-peptide antiserum reacts with the 55K CD3-associated protein on PEER. Most previously described human Ti gamma-chain complementary DNA clones encode the products of non-functional rearrangements. One of the Ti gamma cDNAs isolated from PEER, however, represents a functional rearrangement reported for the first time in a cell which expresses a Ti gamma-chain protein product on the cell surface. Interestingly, a 48-base-pair (bp) sequence in the constant (C) region domain of this functional Ti gamma-chain cDNA is triplicated in PEER and duplicated in other cDNAs isolated from PEER and other cell lines.     

A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.     

The nuclear lamina is a meshwork of intermediate-type filaments

The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane, is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromatin. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three immunologically related polypeptides of relative molecular mass (Mr) 60,000-75,000 (60-70K) termed lamins. Three lamins (A, B, and C) are typically present in mammalian somatic cells. Previous studies on nuclear envelopes of rat liver and Xenopus oocytes suggested that the lamina has a fibrillar or filamentous substructure. Interestingly, protein sequences recently deduced for human lamins A and C from complementary DNA clones indicate that both of these polypeptides contain a region of approximately 350 amino acids very similar in sequence to the coiled-coil alpha-helical rod domain that characterizes all intermediate-type filament (IF) proteins. Here we analyse the supramolecular organization of the native nuclear lamina and the structure and assembly properties of purified lamins, and show that the lamins constitute a previously unrecognized class of IF polypeptides.