Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.     

Transposon Tn4556 of Streptomyces fradiae: nucleotide sequence of the ends and the target sites.

A transposon, Tn4556, has recently been isolated from Streptomyces fradiae (S.-T. Chung, J. Bacteriol. 169:4436-4441, 1987). The ends of Tn4556 were found to contain inverted repeats of 38 base pairs with 70% sequence identity with the ends of Tn3. Insertion of Tn4556 into a Streptomyces plasmid resulted in a 5-base-pair duplication of the target site.     

Distinction between glycoprotein IIIa and the 100-kDa membrane protein (aggregin) mediating ADP-induced platelet activation

Previous studies from our laboratories showed that 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) inhibits ADP-induced platelet shape change, aggregation, and exposure of fibrinogen sites while covalently binding to 100-kDa platelet membrane protein (aggregin) on the intact platelet. Chymotrypsin digests aggregin to a fragment of 70 kDa, abolishing the inhibition, and also cleaves platelet glycoprotein IIIa (GPIIIa) (100 kDa) to a 70-kDa fragment containing the P1A1 epitope. We questioned whether these platelet membrane proteins were distinct. Both 5'-p-[3H]sulfonylbenzoyl adenosine (SBA)-labeled aggregin and 125I-GPIIIa were precipitated by polyclonal antibodies to a 100-kDa fraction of platelet membranes, but aggregin was not precipitated by a monospecific antibody to P1A1 which precipitates GPIIIa. Further a monospecific polyclonal antibody to immunopurified GPIIIa coupled to protein A-Sepharose adsorbed GPIIIa but not aggregin. Similarly, both aggregin and GPIIIa were precipitated by a polyclonal antibody to an isolated 70-kDa component of platelet membrane but only GPIIIa was precipitated by the monoclonal antibody to GPIIIa, (SSA6). Two patients with Glanzman's thrombasthenia whose platelet membranes contained less than 5% GPIIIa as assayed by monoclonal antibody binding (A2A6), incorporated [3H]SBA to the same extent as normal individuals. Furthermore, FSBA inhibited ADP-induced shape change with a similar concentration dependence for both thrombasthenic and normal platelets. Finally, mobility of GPIIIa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was decreased following reduction with dithiothreitol whereas that of [3H]SBA-labeled MP 100 was not altered. We conclude that GPIIIa and aggregin are distinct platelet membrane proteins.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

Further characterization and chemical purity assessment of the human erythrocyte glucose transporter preparation

Chemical and functional purity of the human erythrocyte glucose transporter preparation obtained by DEAE column chromatography after octyl glucoside solubilization was assessed. The cytochalasin B binding capacity of the preparation indicates that the preparation is 60-85% functional glucose transporter. Gel filtration chromatography on TSK 250 column separates this preparation into at least three major peptide fractions, namely, P0, P1 and P2, with apparent Mr of approx. 80 000, 43 000 and 17 000, respectively. When the preparation is photolabelled with [3H]cytochalasin B prior to the separation only P0 and P1 are labelled. Exposure of the preparation to octyl glucoside or to ultraviolet light irradiation results in an increase in P0 in a time-dependent manner with a concomitant and proportional reduction in P1, without affecting P2 appreciably. For individual preparations, relative abundance of P0 and P1 vary widely in a reciprocal fashion, while that of P2 is practically fixed at approx. 10% of the total protein. The specific activity of cytochalasin B binding of each preparation correlates linearly with the relative abundance of P1 of the preparation, which gives a calculated specific binding activity of 22 nmol/mg protein for this fraction. These results indicate that P1 and P0 are native and denatured transporter, respectively, while P2 is contaminating protein impurities. These results demonstrate that the glucose transporter preparation contains approx. 10% of nontransporter protein impurities, with a varying amount (up to 30%) of denatured transporter, and that the transporter free of the chemical impurities and the denatured transporter can be obtained by a gel filtration chromatography of this preparation.     

A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin

A new method for specifically staining the iron atoms present in transferrin and lactoferrin after polyacrylamide gel electrophoresis and isoelectric focusing is described. The stain, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium salt, or Ferene S, will detect transferrin in 5 microliters of human serum, lactoferrin in 10 microliters of human whey, and 10 micrograms of purified primate (Macaca fascicularis) transferrin. This method of staining is very rapid as the serum transferrin bands can be seen within 5 to 10 min of staining.     

Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.     

beta-Internexin, a ubiquitous intermediate filament-associated protein

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.