A Neurofilament-Associated Kinase Phosphorylates Only a Subset of Sites in the Tail of Chicken Midsize Neurofilament Protein

Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher-molecular-mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament-associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle-molecular-mass neurofilament subunit, NF-M. Two-dimensional phosphopeptide mapping of ³²P-labeled NF-M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF-M is used as an in vitro substrate. Phosphopeptide maps from in vitro-phosphorylated NF-M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF-M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF-M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF-M and (b) that multiple kinases are necessary for complete phosphorylation of the NF-M tail.     

A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism.

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.     

Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.     

Reactions Catalyzed by Peg-Modified α-Chymotrypsin in Organic Solvents. Influence of Water Content and Degree of Modification

-Chymotrypsin was modified with cyanuric chloride activated monomethoxypolyethylene glycol (MPEG) with molecular weights 1900 and 5000. Using the higher molecular weight MPEG a product that was soluble in benzene at moderate levels of modification was obtained, whereas with MPEG 1900 almost all the enzyme's amino groups had to be modified for dissolving the conjugate. The catalytic activity decreased with increasing degree of substitution. Apparent V_max was considerably higher for the less modified enzyme preparation than for the more modified one, while K_m,app stayed almost constant. The modified enzyme was used for peptide synthesis. The reaction was dependent on the content of dissolved water. Both V_max,app and K_m,app increased with increasing water content. It was possible to achieve a process with complete conversion of substrate to dipeptide.     

Immunolocalization of the G protein alpha subunit encoded by the GPA1 gene in Arabidopsis.

Heterotrimeric GTP binding proteins (G proteins) are important signal transducers in lower eukaryotes and in animal cells. In plants, the occurrence of GTP binding proteins has been reported, but their biological function remains unclear. Two genes coding for G protein alpha subunits have been cloned: GPA1 in Arabidopsis and TGA1 in tomato. To gain some insights into the function of GPA1, we describe an extensive immunolocalization of GPalpha1, the gene product of GPA1, during Arabidopsis development. Our results show that the GPalpha1 is present through all stages of development and in all organs examined, with the exception of mature seeds. It is expressed in roots, floral stem, rosette leaves, cauline leaves, flowers, and seed pods. Interestingly, the level of GPalpha1 protein is higher in immature organs than in mature organs. GPalpha1 is present at a high level in the root meristem and elongation zone, in the shoot and floral meristems, and in the leaf primordium and floral organ (sepal, petal, stamen, and gynoecium) primordia. During flower development, dividing microspores, but not mature pollen, show high levels of GPalpha1. During pollination, GPalpha1 is present in the growing pollen tubes. The protein is also present in nectaries and developing ovules and, after fertilization, in developing embryos. In mature tissue, GPalpha1 is preferentially found in the vascular system but is also present in other cell types. The complexity of the GPalpha1 localization pattern suggests that GPalpha1 might be involved in different signaling pathways depending on the developmental stage.     

Response regulation in bacterial chemotaxis

The signal transduction system that mediates bacterial chemotaxis allows cells to moduate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue-at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY. © 1993 Wiley-Liss, Inc.     

Feeding Response of Rats to No-Fat and High-Fat Cakes

The nutritional effects of high-fat diets have been extensively studied in laboratory animals, but as yet few experiments have examined the feeding response of animals to newly developed fat substitutes. The present study used commercially available no-fat (0% fat, 92% carbohydrate) and high-fat (41% fat, 54% carbohydrate) cake to determine the effects of fat substitutes on food preference and caloric intake in rats. The first experiment showed that nondeprived rats found the high-fat and no-fat cakes equally palatable and highly preferred to lab chow. Food deprived rats, however, preferred the high-fat cake to the no-fat cake, which may be related to its higher caloric density. In the second experiment, rats fed high-fat cake, in addition to chow, for 30 days consumed more calories and gained more weight than did rats fed no-fat cake and chow. The no-fat cake group, however, overate and gained more weight than chow-only controls. The hyperphagic response to the no-fat cake can be attributed to its carbohydrate content, moisture, and high palatability. Thus, removing fat from the cake reduced, but did not eliminate, its obesity-promoting effect. Obviously, low-fat foods must be consumed in moderation if used for weight control.