Modulation of protein kinases and phosphoprotein phosphatases by a small acidic protein from bovine brains

A small, acidic and heat-stable protein was purified from bovine brains by column chromatography on DEAE-cellulose, Bio-Gel HTP, Affi-Gel phenothiazine and Sephadex G-75. This protein stimulates megamodulin-dependent protein kinase I from brains and phosphoprotein phosphatases from either brain or yeast. However, it inhibits cyclic AMP-dependent protein kinases from skeletal muscle.     

Purification of a membrane protein modifying the sensitivity of Na+/K+ ATPase to ouabain from a supernatant of plasma membrane treated with EDTA]

A membrane protein of M. W. 32,000 D and pI 5 has been purified from EDTA-treated plasma membrane supernatants by Page without detergents. These membranes are purified from the MF2S cell line issued from the murine plasmacytoma MOPC 173. The purified protein was able to induce a 300 fold increase in the Na+/K+ ATPase resistance to ouaba?n thus mimicking the original resistance of the enzyme to the drug.     

Modulation of protein kinases and phosphoprotein phosphatases by a small acidic protein from bovine brains

Summary A small, acidic and heat-stable protein was purified from bovine brains by column chromatography on DEAE-cellulose, Bio-Gel HTP, Affi-Gel phenothiazine and Sephadex G-75. This protein stimulates megamodulin-dependent protein kinase I from brains and phosphoprotein phosphatases from either brain or yeast. However, it inhibits cyclic AMP-dependent protein kinases from skeletal muscle.Acknowledgments. This work was supported by a grant (RR-08229) from the National Institutes of Health, USA. W.N. Kuo is a recipient of a Distinguished Faculty Scholar Award from United Negro College Fund, Inc., USA.     

Use of a zonal rotor for demonstrating that human myelin is formed by a continuum of particles of different densities]

Human myelin isolated from frozen brain consists of a continuum of particles as shown by sucrose continuous gradient (between 0,4 and 0,85 M) on zonal rotor. This was determined by measuring the O. D. at 260 and 280 millimicron, the protein concentration by Lowry method and sucrose concentration by polarimetry in 60 fractions. Most of the material is concentrated between 20-23% sucrose, the maximum being found at 21.5% (0.63 M). The fractions are dialyzed for 62 hrs. to remove the sucrose and the pellet obtained after centrifugation shows that the protein concentration increases from the ligher fraction to the heavier.     

Choice of a spin label probe in order to study the recombination of beta-hydroxybutyrate dehydrogenase of a rat liver inner mitochondrial membrane with lecithin vesicles]

We find from studying the inhibitory effect of N-ethylmaleimide (NEM) on the enzymatic activity of beta-hydroxybutyrate dehydrogenase, that approximately one molecule of NEM is bound for one molecule of protein when the enzymatic activity is completely inhibited. Since the protein is a dimer this implies that each molecule of protein possesses only one thiol group in its catalytic center. Two long chain maleimide derivates: (10.3) NEM and (1.14) NEM conform, if a reasonable assumption is accepted to the conditions required for the study of the recombination of beta-hydroxybutyrate dehydrogenase with lecithin vesicles by spin label technique.     

Nonspecific reaction of a thiol:Protein disulfide oxidoreductase with the disulfide bonds of insulin

Summary A thiol:protein disulfide oxidoreductase from bovine liver was isolated after separation from protein disulfide isomerase. The enzyme, after activation (reduction) with glutathione, was reacted with stoichiometric amounts of insulin and the sulfhydryl groups of the partially reduced hormone were labeled with iodo (l-¹⁴C)acetamide. After separation of the insulin chains, the radioactivity was found in both the peptides, with a ratio A-chain/B-chain equal to 2/1.     

Nonspecific reaction of a thiol: protein disulfide oxidoreductase with the disulfide bonds of insulin

A thiol: protein disulfide oxidoreductase from bovine liver was isolated after separation from protein disulfide isomerase. The enzyme, after activation (reduction) with glutathione, was reacted with stoichiometric amounts of insulin and the sulfhydryl groups of the partially reduced hormone were labeled with iodo (l-14C)acetamide. After separation of the insulin chains, the radioactivity was found in both the peptides, with a ratio A-chain/B-chain equal to 2/1.     

Demonstration of a specific androgen binding protein (ABP) in the seminal plasma of the ram]

A specific androgen binding protein has been demonstrated in the seminal plasma of adult Ram. This protein binds especially to 5 alpha-DHT and testosterone and much lower to oestradiol-17 beta. Its characteristics such as Ka (in order 10(9) M(-1) at 4 degrees C), relative mobility (Rf) and its specificity are similar to those of the androgen binding protein (ABP) of the Rete Testis Fluid and the epididymal plasma of the Ram. It is probable that this protein secreted from the testis, crosses the epididymis before being secreted in the seminal plasma at the moment of the ejaculation.     

The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP^C). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP^C. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP^C-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrP^C signaling.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Cyanovirin-N: a sugar-binding antiviral protein with a new twist

Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N. RID="*" ID="*"Corresponding author.     

Glutamate synthase: a complex iron-sulfur flavoprotein

Glutamate synthase is a complex iron-sulfur flavoprotein that forms l-glutamate from l-glutamine and 2-oxoglutarate. It participates with glutamine synthetase in ammonia assimilation processes. The known structural and biochemical properties of glutamate synthase from Azospirillum brasilense, a nitrogen-fixing bacterium, will be discussed in comparison to those of the ferredoxin-dependent enzyme from photosynthetic tissues and of the eukaryotic reduced pyridine nucleotide-dependent form of glutamate synthase in order to gain insight into the mechanism of the glutamate synthase reaction. Sequence analyses also revealed that the small subunit of bacterial glutamate synthase may be the prototype of a novel class of flavin adenine dinucleotide- and iron-sulfur-containing oxidoreductase widely used as an enzyme subunit or domain to transfer reducing equivalents from NAD(P)H to an acceptor protein or protein domain. Received 10 November 1998, received after revision 10 December 1998; accepted 10 December 1998     

BAG-6, a jack of all trades in health and disease

BCL2-associated athanogene 6 (BAG-6) (also Bat-3/Scythe) was discovered as a gene product of the major histocompatibility complex class III locus. The Xenopus ortholog Scythe was first identified to act as an anti-apoptotic protein. Subsequent studies unraveled that the large BAG-6 protein contributes to a number of cellular processes, including apoptosis, gene regulation, protein synthesis, protein quality control, and protein degradation. In this context, BAG-6 acts as a multifunctional chaperone, which interacts with its target proteins for shuttling to distinct destinations. Nonetheless, as anticipated from its genomic localization, BAG-6 is involved in a variety of immunological pathways such as macrophage function and T_H1 response. Most recently, BAG-6 was identified on the plasma membrane of dendritic cells and malignantly transformed cells where it serves as cellular ligand for the activating natural killer (NK) cell receptor NKp30 triggering NK cell cytotoxicity. Moreover, target cells were found to secrete soluble variants of BAG-6 and release BAG-6 on the surface of exosomes, which inhibit or activate NK cell cytotoxicity, respectively. These data suggest that the BAG-6 antigen is an important target to shape a directed immune response or to overcome tumor-immune escape strategies established by soluble BAG-6. This review summarizes the currently known functions of BAG-6, a fascinating multicompetent protein, in health and disease.     

Surface display of a single-domain antibody library on Gram-positive bacteria

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10⁷ camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.     

A vector system designed to allow excretion of a protein encoded by a cloned gene]

We describe a method which should allow construction of bacterial strains able to export a given protein through the cytoplasmic membrane. The principle is to fuse the structural gene of the protein to the proximal part of gene lamB, the structural gene of the lambda receptor, an outer membrane protein of E. coli K-12.     

Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship

A direct relationship was established between the stability of the lysosomal membrane and an estimate of cytosolic protein catabolism, based on loss of radiolabel from prelabeled protein. Lysosomes in the lysosomally-rich digestive cells of the midgut gland of the marine mussel (Mytilus edulis) were destabilized by experimental treatment with phenanthrene.     

Expressed protein ligation: a resourceful tool to study protein structure and function

This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review, we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function.     

Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship

Summary A direct relationship was established between the stability of the lysosomal membrane and an estimate of cytosolic protein catabolism, based on loss of radiolabel from prelabeled protein. Lysosomes in the lysosomally-rich digestive cells of the midgut gland of the marine mussel (Mytilus edulis) were destabilized by experimental treatment with phenanthrene.     

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold

The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolu tionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space. Received 9 October 1997; received after revision 6 January 1998; accepted 19 January 1998