Properties of soluble somatostatin-binding protein

A soluble somatostatin-binding protein was detected in the cytosol fractions of various rat, human and bovine tissues. Maximum binding occurred at pH8.0-8.5 and was Ca(2+)-dependent. The specific binding of somatostatin per 10mug of cytosol protein from 12 rat tissues ranged between 36 and 15%, and 3% for peripheral blood cells. There was also substantial binding in cytosol from human anterior pituitary and liver, and bovine anterior pituitary. The specific binding in rat and human plasma in the presence of EDTA was only 1%. Gel filtration suggested a molecular weight of approx. 80000 for the somatostatin-binding protein from several sources. Exposure of the binding protein to trypsin eliminates somatostatin-binding activity but ribonuclease and deoxyribonuclease have no effect. The binding protein is thermolabile, ethanol-precipitable, and not completely specific for somatostatin. Bound (125)I-labelled [Tyr(1)]somatostatin is not easily displaced by excess of unlabelled somatostatin. The effects of dithiothreitol and mercaptoethanol on the binding of (125)I-labelled [Tyr(1)]somatostatin to the binding protein suggests that binding involves two sequential steps, first loose binding, then disulphide linkage. Since semipurified somatostatin-binding protein causes a dose-related inhibition of the binding of (125)I-labelled [Tyr(1)]somatostatin in radioimmunoassays for somatostatin, estimates of somatostatin content of tissue extracts by radioimmunoassay in some cases may be spuriously high. It is not yet clear whether the binding protein is a true cytosol protein or an easily solubilized membrane protein.     

Ultracentrifugation evidence for a somatostatin-binding protein in serum.

Using a technique of high speed centrifugation of serum and a well validated immunoassay for the measurement of serum somatostatin-like immunoreactivity, we have demonstrated that somatostatin, unlike other peptide hormones, appears to sediment with large molecular weight proteins. When synthetic somatostatin of increasing concentration was incubated with serum prior to ultracentrifugation, a linear plot of concentration of somatostatin added against concentration sedimenting (or apparently bound to protein) revealed an association curve. These data provide further evidence for the existence of a serum-binding protein for somatostatin.     

Specific somatostatin-binding to cytosol of bovine gallbladder mucosa

The binding of somatostatin was studied in the cytosolic fraction of bovine gallbladder mucosa. The binding reaction depended on time, temperature and pH, and was reversible, saturable and specific. Stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (K _d=23.6 nM) and low capacity (3.7 pmol somatostatin/mg protein) and a class with low affinity (K _d=284.6 nM) and high capacity (85.0 pmol somatostatin/mg protein) at 37°C and pH 7.4. The binding sites were highly specific for somatostatin since peptides such as [Leu]enkephalin, neurotensin, vasoactive intestinal peptide and substance P showed practically no effect upon somatostatin binding. The presence of somatostatin-binding sites in the cytosolic fraction of gallbladder mucosa, together with the known occurrence of somatostatin nerve endings in the gallbladder strongly suggests that this peptide may be involved in the physiology and physiopathology of gallbladder mucosa.     

Characteristics of a de novo designed protein.

A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements. The GuHCl denaturation is well described by a 2-state model. The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.     

Characteristics of a nuclear protein kinase from rat epididymis

Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)₂SO₄. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg²⁺, Co²⁺, Ca²⁺ or Mn²⁺. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co²⁺)-dependent enzyme. The apparentK _m values of the enzyme for casein, ATP and Co²⁺ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine. The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized.     

Characteristics of a high molecular weight extracellular protein of Streptococcus mutans

A high molecular weight protein antigen, designated P1, has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectrophoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B, I/II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues, particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1, when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Characteristics of C6 glioma cells overexpressing a gap junction protein

1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 mRNA and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 mRNA did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling.     

Characteristics of HIV-Tat protein transduction domain

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study examined the intercellular transport and protein transduction of the Tat protein. The results showed no evidence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This suggests that the HIV-1 PTD passes through the plasma membrane in one direction.     

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.     

Characteristics of protein splicing in trans mediated by a semisynthetic split intein

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.     

抗冻蛋白的特性和作用机制

抗冻蛋白是非常热门的研究领域,具有广阔的应用前景。本文结合最新的报道,从抗冻蛋白的多样性、热带活性和改变冰的生长,综述了抗冻蛋白的特性,阐释了抗冻蛋白的作用机制的三种假说：“偶极子—偶极子”假说、氢原子结合模型假说和刚体能量学说。     

Characteristics of protein-storing cells associated with a 67 kDa protein in Hevea brasiliensis

 The protein-storing cells (PSCs) in Hevea brasiliensis were studied by using light- and electron-microscopy and SDS-PAGE. The cells were found in stem and root where secondary phloem was developed. They are a special kind of phloem parenchyma cell which accumulate in their central vacuoles large amounts of protein, fibril-like under an electron microscope, and have few plastids with very small starch grains. Their distribution is strictly restricted to the secondary phloem axial system where they exactly sequestered in functional phloem or slightly over it. A 67 kDa protein was always found in the tissues where the PSCs were observed. During the first seasonal growth flush, the 67 kDa protein in the terminal branchlet exhibits marked quantitative fluctuation which is consistent with the change of the vacuole protein inclusion of the PSCs in the branchlet. These facts suggested that the 67 kDa protein might be the major part of the vacuole protein of the PSCs. Considering the differences between the PSCs in Hevea and the PSCs in the other trees studied, we define two types of PSCs: Hevea-type, which are the cells specialized for protein storage and Populus-type, which are ordinary parenchyma cells accumulating protein and starch. Received: 11 April 1997 / Accepted: 28 July 1997     

Characteristics of extracellular protein production by a plasmidless derivative of Staphylococcus simulans biovar staphylolyticus

A derivative of Staphylococcus simulans biovar staphylolyticus cured of all five plasmids present in the wild-type organism was developed, and the characteristics of extracellular protein production by this plasmidless strain were compared to those of the wild type. Although staphylolytic endopeptidase (lysostaphin) and beta-lactamase are known to be plasmid encoded, analysis of this cured strain revealed that most other extracellular proteins are chromosomally encoded.     

Characteristics of thiamine-binding protein from rat brain synaptosomes

The thiamine-binding protein was obtained from rat brain synaptosomes by affinity chromatography and gel-filtration on Sephadex G-200. The protein is homogeneous by the data of SDS gel-electrophoresis, anode electrophoresis and isofocusing between pH 3.5-9.0. The isoelectric point of this protein is near pH 4.8-5.0. The binding nature of the protein with [14C] thiamine was studied. It is shown that metal ions, especially Na+ and Ca2+, increase the thiamine-binding activity. The binding process is of a saturation character at the thiamine concentrations of 10(-7)-10(-5) M. Thiamine possesses two binding sites with KD1 = 3.1 microM and KD2 = 30 microM. Out of the tested thiamine analogues and antagonists of vitamin B1 thiamine-monophosphate and pyrithiamine were the most competitive.