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We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates. [ABSTRACT FROM AUTHOR]

Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 ± 0.2 μ m. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 ± 0.2 μ m and a kcat of 0.23 ± 0.004 s−1, respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. Structured digital abstract ● and by () ● by () [ABSTRACT FROM AUTHOR]

Periplasmic membrane-bound [NiFe]-hydrogenases undergo a complex maturation pathway, including cofactor incorporation, subunit assembly, and finally twin-arginine-dependent membrane translocation (Tat). In this study, the role of the two accessory proteins HoxO and HoxQ in the maturation of the membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha H16 was investigated. MBH activity was absent in soluble as well as membrane fractions of cells with deletions in the respective genes. The absence of HoxO and HoxQ led to degradation of the small subunit precursor (preHoxK) of the MBH. The two accessory proteins directly interacted with preHoxK prior to assembly of active MBH dimer in the cytoplasm. MBH mutants with modified Tat signal peptides were disrupted in preHoxK/HoxO/HoxQ complex formation. Isolated HoxO and HoxQ proteins formed a complex in vitro with the chemically synthesized HoxK Tat signal peptide. Two functions of the two chaperones are discussed: (i) protection of the Fe–S cluster containing HoxK subunit under oxygenic conditions, and (ii) avoidance of HoxK export prior to dimerization with the large MBH subunit HoxG. [ABSTRACT FROM AUTHOR]

Problem The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. Methods of study The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. Results ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. Conclusions Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model. [ABSTRACT FROM AUTHOR]

The diffraction pattern of native protein crystals of F420-dependent methylenetetrahydro-methanopterin dehydrogenase from Methanopyrus kandleri shows weak additional reflections compared with the selenomethionine-labelled protein crystals, indicating a doubled c unit-cell parameter. These reflections indicate small reorientations of the hexameric structural units, breaking the translational symmetry. TLS refinement of the selenomethionine-labelled protein structure at 1.55 Å resolution revealed an anisotropic rigid-body libration of the hexameric units. The anisotropy is consistent with the static reorientation in the native protein crystals. These results are discussed as related to the crystal packing. The relation between the two structures suggests an analogy to structural changes during certain kinds of phase transitions that have been well studied in inorganic structural chemistry. [ABSTRACT FROM AUTHOR]

Research on the export of proteins and nucleic acids from the nucleus to the cytoplasm has greatly gained from the discovery that the actinobacterial toxin leptomycin B (LMB) specifically inactivates the export receptor chromosomal region maintenance 1 (CRM1). Recently, it was shown that myxobacterial cytotoxins, named ratjadones (RATs), also bind to CRM1 and inhibit nuclear export. However, the reaction mechanism of RATs was not resolved. Here, we show that LMB and RAT A employ the same molecular mechanism to inactivate CRM1. Alkylation of residue Cys528 of CRM1 determines both LMB and RAT sensitivity and prevents nuclear export of CRM1 cargo proteins. [Copyright &y& Elsevier]

Adiponectin is an adipocyte-specific secretory protein which exhibits antiatherogenic, anti-inflammatory and antidiabetic properties. We hypothesized that testosterone plays an important role in the regulation of its secretion in humans, as adiponectin concentrations are higher in women than in men and as testosterone administration is accompanied by a reduction in serum adiponectin in animals and by reduced protein secretion in cultured adipocytes. This study aimed to evaluate adiponectin levels in hypogonadal men prior to and during testosterone replacement therapy. In a retrospective study, adiponectin, total and free testosterone, oestradiol, SHBG, total cholesterol and triglyceride levels were evaluated in 31 hypogonadal men [HM; age, mean ± SEM: 36·5 ± 2·4 years; body mass index (BMI) 24·6 ± 0·8 kg/m2] and 29 weight-matched eugonadal men (EM; age 30·8 ± 1·5 years; BMI 23·4 ± 0·6 kg/m2). In 13 HM (age 33·9 ± 3·2 years; BMI 24·2 ± 0·9 kg/m2) the same parameters were also evaluated after 6 months of testosterone replacement therapy. Correlation analysis between adiponectin and hormonal, biochemical and anthropometric parameters was performed in all subjects. Testosterone, free testosterone and oestradiol concentrations were significantly lower in HM than in EM (4·4 ± 0·4 nmol/l, 78·4 ± 10·9 pmol/l and 36·1 ± 3·0 pmol/l, respectively, in HM vs. 21·9 ± 0·7 nmol/l, 507·9 ± 13·8 pmol/l and 65·2 ± 1·8 pmol/l, respectively, in EM, P < 0·0001), while SHBG levels in HM were higher than in EM (54·4 ± 7·5 vs. 30·9 ± 2·2 nmol/l, P < 0·005). Serum adiponectin levels in HM were significantly higher than in EM (9·53 ± 0·73 vs. 6·80 ± 0·55 µg/ml, P < 0·01). Calculation of the Pearson coefficient showed that adiponectin levels in HM were not correlated with any of the anthropometric and hormonal parameters examined, but showed a significant negative correlation with serum triglycerides ( r = −0·38, P < 0·05). Serum adiponectin levels were negatively correlated with body weight ( r = −0·41, P < 0·05) in EM but not with other anthropometric, hormonal or biochemical parameters. Six months after initiation of testosterone replacement therapy, which increased testosterone and free testosterone levels to the normal range, adiponectin levels were significantly reduced in HM (6·37 ± 0·93 vs. 9·26 ± 1·01 µg/ml, P < 0·01) and similar to those recorded in EM. Compared to eugonadal subjects, hypogonadal men show higher adiponectin levels which are reduced by testosterone replacement therapy. This study indicates that testosterone exerts a regulatory role on adiponectin secretion in humans. [ABSTRACT FROM AUTHOR]

The structure of the 115 amino-acid residue protein DsvC was determined based on the anomalous scattering provided by the five S atoms present in the structure. By collecting the diffraction data at a wavelength of 1.9 Å. the anomalous signal provided by the S atoms was enhanced. However, significant radiation damage occurred during the course of the experiment, which led to differences between different parts of the data set. Only by dividing the total data set into five data sets was it possible to obtain phases; these could then he successfully extended to allow structure determination by the automated model-building program ARP/wARP. A computational correction for the radiation damage was found to significantly improve the success rate in determining the heavy-atom substructure and to improve phasing and refinement statistics. [ABSTRACT FROM AUTHOR]

Background Leucocyte migration within inflammatory skin compartments in allergic contact dermatitis (ACD) is the result of a sophisticated multi-step event where multiple molecules are involved. Objective Since non-antigen-specific mechanisms have been described as an early participant in elicitation of ACD, we investigated the kinetics of the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and the type of infiltrating cells. We compared the time course production of MCP-1/CCL2 with connecting segment-1 (CS-1) fibronectin and thymus and activation-regulated chemokine (TARC/CCL17) expression. Methods Biopsies from 10 individuals challenged in their back with the antigen responsible for their contact dermatitis and an irrelevant antigen were taken at different times and histology, immunohistochemistry for CS-1 fibronectin, TARC/CCL17, CD3, CD68, CXCR3, CCR4 and in situ hybridization for MCP-1/CCL2 were performed. Results At positive antigen stimulated sites expression of MCP-1/CCL2 by basal keratinocytcs and isolated cells in dermis started at 10h. CS-1 fibronectin and TARC/CCL17 expression by blood endothelial cells was found at 2 and 10h, respectively. This was followed by dermal accumulation of mononuclear cells with a significant increase of CD3[sup+] and CD68[sup+] cells. At 48h, approximately 58% of infiltrating cells were CXCR3[sup+], and 35% CCR4[sup+]. Conclusions We showed evidence of the fact that CS-1 fibronectin expression precedes the production of MCP-1/CCL2 and TARC/CCL17 in the skin of patients with ACD, suggesting that these molecules participate in the early complex process of migrating mononuclear cells during elicitation of ACD. [ABSTRACT FROM AUTHOR]

Various functions are ascribed to the HBx regulatory protein of the hepatitis B virus (HBV). Due to the low expression level of HBx, it has been difficult to correlate spatial and temporal HBx expression levels with specific functions. Based on a novel cell-permeable peptide, known as the translocation motif (TLM), cell-permeable HBx fusion proteins were generated. The TLM-HBx fusion protein is rapidly internalized from the medium into almost all cells, whereas no significant internalization was seen with wild-type HBx. The major fraction of internalized HBx protein moves from the cytoplasm to the nucleus. The cytosolic fraction, however, activates c-RAF1/extracellular-signal-related kinase 2 signalling and causes activation of activator protein 1 (AP1) and nuclear factor-κB. The TLM-HBx protein rescues HBV gene expression from an activator-deficient HBV genome. These results indicate that cell-permeable regulatory proteins provide a novel, efficient tool for a clearly defined, dose-dependent analysis of regulatory protein function, without affecting the integrity of the cell, and can be used for the safe reconstitution of virus production from a regulatory-protein-deficient virus genome. [ABSTRACT FROM AUTHOR]

Covalent lipid attachments are essential co- and posttranslational modifications for signalling proteins. Gαs, the α-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Gαs to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly²). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Gαi are downregulated to <10%. Thereby, Gly2-palmitoylation of Gαs relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Gαi more difficult. [ABSTRACT FROM AUTHOR]

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca2+ influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1β (mTRP1β) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1β expressing cells exhibited enhanced Ca2+ influx and enhanced whole-cell membrane currents compared to mTRP1β deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1β is required for structural organization thus forming functional channels. [Copyright &y& Elsevier]

The canalicular multidrug resistance protein 2 (MRP2; gene symbol: ABCC2) mediates ATP-dependent biliary excretion of organic anions such as bilirubin diglucuronide, glutathione conjugates and sulfated and glucuronidated bile salts. In chronic cholestatic liver diseases, the biliary excretion of cholephilic organic anions is impaired. While the underlying transport defects have been studied in rat models of cholestasis, little is known about the molecular basis of impaired organic anion excretion in human cholestatic liver disease. Our aim, therefore, was to analyze expression of MRP2 in patients with primary biliary cirrhosis (PBC), a chronic cholestatic liver disease characterized by progressive destruction of small intrahepatic bile ducts. Four patients with PBC stages III (n=1) and IV (n=3) were compared with three non-cholestatic patients with alcoholic liver disease, idiopathic liver cirrhosis and cirrhosis from chronic hepatitis C. Immunohistochemistry was performed on paraffin-embedded tissue slides using a monoclonal antibody to MRP2. MRP2 was detected at the canalicular hepatocyte membrane of all patients. In two PBC patients (stages III and IV, respectively), the degree of immunostaining was comparable with controls, whereas in two other PBC patients with stage IV disease immunostaining was decreased. We conclude that MRP2 expression decreases with progressive cholestasis in PBC. [Copyright &y& Elsevier]

Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family. [ABSTRACT FROM AUTHOR]

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase). MobB and MobC (Scherzinger, E., Lurz, R., Otto. S & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-481. In this study we report the purification of MobA to apparent bomogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by sing nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex. [ABSTRACT FROM AUTHOR]

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition C2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine α-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of α-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the α-lactalbumin-Ca2+ complex in 50 mM Hepes containing 150 mM Na2+ at pH 7.4 and 25 °C, was found to be 123 ± 2 nM and 103 ± 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to α-lactalbumin did not depend on pH in the range 6.6–8.4 and was differently affected by Na2+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na2+ at pH 7.4, was lowered to 119 ± 13 nM and the number of Ca2+ bound/molecule α-lactalbumin was lowered to 0.069 ± 0.006. No interaction between fluo-3 and α-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements. [ABSTRACT FROM AUTHOR]

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell cybernetic aspects. [ABSTRACT FROM AUTHOR]

The T 7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initiate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involves the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3' : 5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CTP are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initiators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2-4-fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from initiation of DNA chains tie novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA- binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with φX174 DNA as template leads to the generation of 'rolling circle-dike' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initiations can occur tie novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo. [ABSTRACT FROM AUTHOR]

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zürich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of ΔH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to AH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-α chains and NO-Hb Zürich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-β chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-β chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the α chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened σ-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure. [ABSTRACT FROM AUTHOR]

The light-stable tobacco phytochrome apoptrotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct. The spectral properties of the reconstituted PHYB-phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies. The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far-red-light-absorbing (Pfr) form and 356 nm and 658 nm for the red-light-absorbing (Pr) form. The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB-phycocyanobilin adduct with the spectral properties of the Pr form. Kinetic analyses of the in vitro reconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first-order rate constant of 2.8 × 10−²s−¹. The similarity with the reported rate constant for the reconstitution of light-labile phytochrome (PHYA) from oat [Li, L. & Lagarias, J. C. (1992) Phytochrome assembly. J. Biol. Chem. 267, 19 204–19 210] suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB. [ABSTRACT FROM AUTHOR]

Examines the structure of DNA junctions and their interaction with enzymes. Cruciform structures formed by inverted-repeat sequences in supercoiled DNA; Attempts to model the structure of a four-way junction in DNA; Analysis of the structure of the four-way junction by fluorescence resonance energy transfer and gel-electrophoretic analysis.

Hepatic-type fatty-acid-binding protein (hFABP) from the cytosol of bovine liver is a 14.4-k Da neutral protein with a blocked N-terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 µM and 2.15 µM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12% α-helix, 45% β-structure, 15% β-turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as ¹H-NMR analysis of His-C2 and His-C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyro-carbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From ¹H-NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/ protein interaction. [ABSTRACT FROM AUTHOR]

Secretion of hemopexin (20% carbohydrate) and its dependence on glycosylation was studied in primary rat hepatocyte cultures in comparison to the secretion of transferrin (5% carbohydrate). 1. In pulse-chase experiments with [35S]methionine half of the labeled hemopexin was secreted in 30 min. By contrast, it took approximately 50 min for secretion of half of the transferrin. 2. Tunicamycin treatment of cultures significantly delayed the secretion of hemopexin but not that of transferrin. 3. During the pulse period a prominent intracellular precursor of hemopexin, smaller than the mature protein, was evident. It is concluded that the extent of glycosylation of a secretory protein is not necessarily a determinant of the transit time required for intracellular processing and secretion. In the case of hemopexin the glycosylation apparently facilitates the secretion although it is not an absolute prerequisite for the exocytosis of this protein. [ABSTRACT FROM AUTHOR]

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite. [ABSTRACT FROM AUTHOR]

The properties of proteolytically non-modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be s200.w = 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found. The partial specific volume of the enzyme as determined by three different methods at 20 °C is v2 = 0.742 ml/g. From equilibrium sedimentation a molecular weight of 835000 + 32000 was calculated for the native enzyme. Amino acid analysis of the enzyme was performed and the number of half-cystine residues determined as 11⁄100000g is in good agreement with the number of titratable -SH groups after denaturation of the enzyme. The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15°/, smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate. Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered. [ABSTRACT FROM AUTHOR]

The sixth (C′6), seventh (C′7) and eighth (C′8) components of human complement were separated from each other by chromatography on hydroxyl apatite and obtained in functionally pure form. Density gradient ultracentrifugation revealed that C′6 and C′7 interact with the fifth component of complement (C′5) to form a reversible protein-protein complex. Evidence was obtained that this complex occurs also in unfractionated, fresh human serum. Kinetic analysis of the reaction sequence in immune haemolysis disclosed that C′5, C′6 and C′7 act, in this order, subsequent to the third component (C′3) and prior to C′8. Exploration of the mode of action of C′5, C′6 and C′7 led to the formulation of two alternative hypotheses. The three components act either sequentially or as one functional unit. Their fluid phase interaction, the shape of dose-response curves and the encountered difficulty to isolate intermediate reaction products favour the concept that they act as a functional unit. [ABSTRACT FROM AUTHOR]

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps. Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity. [ABSTRACT FROM AUTHOR]