The refined 2.3 A crystal structure of human leukocyte elastase in a complex with a valine chloromethyl ketone inhibitor

The stoichiometric complex formed between human leukocyte elastase and a synthetic MeO-Suc-Ala-Ala-Pro-Val chloromethyl ketone inhibitor was co-crystallized and its X-ray structure determined, using Patterson search methods. Its structure has been crystallographically refined to a final R value of 0.145 (8.0 and 2.3 A). The enzyme structure is very similar to that recently observed in a complex formed with the ovomucoid third domain from turkey [(1986) EMBO J. 5,2453-2458]. The rms deviation of all alpha-carbon atoms is 0.32 A. The peptidic inhibitor is bound in a similar overall conformation as the ovomucoid binding segment. Covalent bonds are formed between Val-P1 of the inhibitor and His-57 NE2 and Ser-195 OG of the enzyme. The carbonyl carbon is tetrahedrally deformed to a hemiketal. The valine side chain is arranged in the S1 pocket in the g-conformation.     

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.     

Contribution of macrophages to immediate hypersensitivity reaction

The interaction of mast cells with other leukocytes during immediate hypersensitivity reactions was tested by in vivo and in vitro experiments. Intraperitoneal challenge of passively sensitized rats with antigen caused the production of peptidoleukotrienes, leukotriene (LT)B4, thromboxane (TX)B2, and 6-keto-prostaglandin (PG) F1 alpha in the peritoneal cavity. Pretreatment of the rats with thioglycollate i.p. markedly changed the amount of eicosanoids formed. When polymorphonuclear leukocytes were the predominant cell type in the peritoneal exudate, both LTC4 and 6-keto-PGF1 alpha were decreased by 75% each and TXB2 by 50%. When elicited macrophages were predominant, there was an additional reduction in LTC4 by 68% as compared with 18 hr after thioglycollate treatment, but no additional change in the other arachidonic acid metabolites. In vitro antigen challenge of passively sensitized mouse bone marrow-derived mast cells caused the release of LTC4, LTB4, 6-trans-LTB4, 5-hydroxyeicosatetraenoic (5-HETE), and TXB2. Exposure to antigen of these mast cells in the presence of resident peritoneal macrophages markedly altered eicosanoid formation. Early in the time course (2 to 15 min), macrophages markedly enhanced all 5-lipoxygenase products. However, later in the time course (30 to 120 min), these products were decreased. This decrease was reversed by catalase and superoxide dismutase, which suggests the involvement of oxygen radicals. These active oxygen species also seemed to be generated by mast cells, because these enzymes caused an increase in 5-lipoxygenase products when mast cells were challenged alone. RIA of cyclooxygenase products showed that mast cells released only TXB2 when stimulated with antigen. When they were stimulated in the presence of macrophages, TXB2 and also PGE2 and 6-keto-PGF1 alpha were synthesized. Therefore, macrophages probably contribute the PGE2 and 6-keto-PGF1 alpha. Because the same amount of TXB2 was generated whether macrophages were present or not, the mast cells seem to be the major source of this compound. These data indicate that macrophages and possibly polymorphonuclear leukocytes participate in immediate hypersensitivity reactions.     

Degradation of Dehydrodivanillin by Anaerobic Bacteria from Cow Rumen Fluid

Dehydrodivanillin (DDV; 0.15 g/liter) was biodegradable at 37°C under strictly anaerobic conditions by microflora from cow rumen fluid to the extent of 25% within 2 days in a yeast extract medium. The anaerobes were acclimated on DDV for 2 weeks, leading to DDV-degrading microflora with rates of degradation eight times higher than those initially. Dehydrodivanillic acid and vanillic acid were detected in an ethylacetate extract of a DDV-enriched culture broth by thin-layer, gas, and high-performance liquid chromatographies and by mass spectrometry.     

Chemical transformation of eremofortin C into PR toxin.

The natural products of both eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Because the chemical structures of EC and PR toxin are closely related to each other and differ only by a hydroxyl functional group in EC and an aldehyde functional group in PR toxin at the C-12 position, the chemical transformation of EC into PR toxin was investigated. Oxidation with a chromic anhydride-pyridine complex was found to be the most satisfactory method.     

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.     

Covalent structure of three phase-1 flagellar filament proteins of Salmonella

Using recombinant DNA techniques, the covalent structure was determined for three flagellar filament proteins produced by Salmonella serotypes with phase-1 antigens a, c and d. Comparison of the results obtained, together with previous results for antigen i, indicated an overall structure in which conservation of amino acid sequence was absolute at both ends of the molecule and proceeded inwards with progressively greater variation. Very few differences in nucleotide sequence were detected in regions of amino acid conservation, which suggested that these areas of the gene may be involved in regulatory functions.     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.