Effect of trypsin on the cell surface proteins of hepatoma tissue culture cells. Characterization of a carbohydrate-rich glycopeptide released from a calcium binding membrane glycoprotein.

Concentrations of trypsin that bring about aggregation of hepatoma tissue culture (HTC) cells also release from the cell surface an Mr = 55,000 glycopeptide fragment. This glycopeptide fragment also accumulates in the medium, including serum-free medium, as a normal consequence of membrane protein turnover. The trypsin-released glycopeptide is labeled when cells are grown in the presence of fucose or leucine before treatment of the cells with the protease. Similarly, the glycopeptide fragment can be labeled by reacting cells in situ by lactoperoxidase-catalyzed radioiodination or by tritiated borohydride reduction of cells treated first with neuraminidase and galactose oxidase. The tryptic glycopeptide fragment was purified by concanavalin A-Sepharose chromatography, and hydroxyapatite chromatography in the presence of dodecyl sulfate. The amino acid and carbohydrate composition was determined, as was the sensitivity of the purified glycopeptide to a variety of endo- and exoglycosidases. The purified glycopeptide contains an average of 17 sialic acid residues and hence, shows charge heterogeneity after electrophoresis in isoelectric focusing gels. The charge heterogeneity can be eliminated completely by treatment with neuraminidase. The glycopeptide after this treatment is homogeneous. The trypsin-sensitive membrane glycoprotein which is the source of the Mr = 55,000 glycopeptide was identified by two-dimensional gel electrophoretic analysis of labeled cells, treated or not treated with trypsin. This glycoprotein, which has an apparent molecular weight of 85,000 and forms a homodimer in the presence of calcium ions, was purified and its identity as the parent of the Mr = 55,000 glycopeptide was confirmed by showing that the same Mr = 55,000 fragment was released by trypsin from the purified glycoprotein as was released from the intact cells.     

The application of linear irreversible thermodynamics to biological growth

We measured the rate of cell biomass growth, the consumption of organic substrate, and the heat developed during growth. The relationship between thermodynamic efficiency and the rate of microbial growth was considered.     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.     

Polarity as a criterion in protein design

Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior. Three of the seven hypothetical structures tested here can be rejected as having too many polar side-chain groups in the interior or too few on the protein surface. In addition, a recently designed nutritional protein is identified as being very much unlike globular proteins. These database-derived characteristic quantities are useful in screening designed proteins prior to experiment and may be useful in screening experimentally determined (X-ray, NMR) protein structures for possible errors.     

Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase

Formaldehyde dehydrogenase (EC 1.2.1.1) is a widely occurring enzyme which catalyzes the oxidation of S-hydroxymethylglutathione, formed from formaldehyde and glutathione, into S-formyglutathione in the presence of NAD. We determined the amino acid sequences for 5 tryptic peptides (containing altogether 57 amino acids) from electrophoretically homogeneous rat liver formaldehyde dehydrogenase and found that they all were exactly homologous to the sequence of rat liver class III alcohol dehydrogenase (ADH-2). Formaldehyde dehydrogenase was found to be able at high pH values to catalyze the NAD-dependent oxidation of long-chain aliphatic alcohols like n-octanol and 12-hydroxydodecanoate but ethanol was used only at very high substrate concentrations and pyrazole was not inhibitory. The amino acid sequence homology and identical structural and kinetic properties indicate that formaldehyde dehydrogenase and the mammalian class III alcohol dehydrogenases are identical enzymes.     

A promising genomic transfectant into Xeroderma pigmentosum group A with highly amplified mouse DNA and intermediate UV resistance turns revertant

Following transfection of genomic mouse DNA into an SV40 transformed fibroblast cell line from a patient with Xeroderma pigmentosum (complementation group A, XPA), a single UV resistant cell clone was isolated out of a total of 10(4) independent transfectants. The recipient XPA cell line has as yet not produced spontaneous revertants among 2.2 x 10(8) cells. The isolated cell clone contains 50-70 kb of mouse sequences which are heavily amplified (500-fold), and has acquired both intermediate resistance to UV killing and intermediate unscheduled DNA synthesis (UDS) capacity. By continued passage without selective pressure, cells were generated, which had lost both the dominant marker gene and repetitive mouse sequences. Single colonies of these cells were still intermediately resistant to UV suggesting that either undetected unique mouse DNA had segregated from the bulk of repetitive DNA, or, more likely, that the initially isolated transfectant was a spontaneous revertant. This documents that a persuasive clone isolated can still be a false positive (spontaneous revertant) and that an extremely laborious approach may lead into a dead end.     

Pea aphid symbiont relationships established by analysis of 16S rRNAs

The pea aphid (Acyrthosiphon pisum Harris) harbors two morphologically distinct procaryotic intracellular symbionts. The genes for the 16S rRNA from these symbionts have been cloned and sequenced. Comparisons with sequences of 16S rRNAs from selected procaryotes indicate that the two symbionts are evolutionarily distinct from each other and are members of the gamma-3 subdivision of the class Proteobacteria. One of the symbionts is a member of the family Enterobacteriaceae, while the other constitutes a lineage distinct from these organisms. Both symbionts appear to have only one copy of their rRNA operon.