Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.     

Determination of the microscopic and macroscopic acid dissociation constants of glycyl-L-histidyl-L-lysine and related histidine peptides.

Proton magnetic resonance studies of the acid-base chemistry of the glycyl ammonium, histidyl imidazolium, and lysyl ammonium groups of glycyl-L-histidyl-L-lysine and of the glycyl ammonium and histidyl imidazolium groups of glycyl-L-histidine and glycyl-L-histidylglycine are described. Chemical-shift data indicate that, at the molecular level, the glycyl ammonium and the histidyl imidazolium groups are titrated over the same pH range, with the acidity of the imidazolium group some 8 to 10 times that of the glycyl ammonium group, depending on the peptide. The lysyl ammonium group of Gly-His-Lys is much less acidic and is titrated over a higher pH range. Microscopic and macroscopic acid-dissociation constants were determined from chemical-shift data for each of the peptides. It is shown how microscopic formation constants for protonated metal complexes of these ligands, which are being used increasingly as models for the binding of metal ions by proteins, can be calculated from the macroscopic formation constants and the microscopic acid-dissociation constants. The acid-base chemistry of Gly-His-Lys is discussed with respect to its recently discovered biological activity.     

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.     

Growth factor-initiated proliferation of mouse embryonic fibroblasts induces cytotoxicity by natural killer cells and by a non-cytolysin cytotoxin in natural killer granules

NK cells preferentially kill normal embryonic fibroblasts. Because embryonic cells are growth factor responsive and maintain high proliferative rates, we examined the requirement for growth factor-initiated proliferation for NK susceptibility. Murine embryonic fibroblasts made quiescent in defined medium lacking growth factors were relatively resistant to NK cytolysis. However, reinitiation of proliferation with basic fibroblast growth factor (bFGF) or epidermal growth factor enhanced lysis in a dose-dependent fashion. TGF-beta, which blocked cell division, did not enhance cytotoxicity. Additionally, growth inhibition by prolonged incubation at confluence suppressed lysis. The enhanced NK cytotoxicity of bFGF-stimulated fibroblasts was caused by a post-binding event because no difference in cold target inhibition could be demonstrated with bFGF-treated cells. NK cytotoxicity has largely been attributed to the action of cytotoxins released from cytoplasmic granules. In a 51Cr release assay, bFGF-treated fibroblasts were insensitive to NK granules isolated from the RNK large granular lymphocyte leukemia. However, these same cells exhibited marked sensitivity to lysis in an 18-h adhesion assay normally utilized to detect TNF-alpha. With the use of this assay, a dose-dependent increase in sensitivity of bFGF-treated fibroblasts was observed, whereas quiescent fibroblasts were resistant to the action of isolated NK granules. Granule cytotoxicity was not caused by cytolysin/perforin because inactivation of granule hemolytic activity with CaCl2 did not affect fibroblast killing, and bFGF-treated cells were insensitive to purified cytolysin/perforin. This suggested that another granule associated cytotoxin was responsible for enhanced NK sensitivity of actively proliferating fibroblasts.     

Modification of three enzymes of the β-ketoadipate pathway by N-methyl-N′-nitro-N-mtrosoguanidine

The sensitivities of three enzymes of the β-ketoadipate pathway to inactivation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were determined in vivo and in vitro under conditions compatible with mutagenesis.One enzyme, β-ketoadipate enol-lactone hydrolase, is very sensitive to inactivation by low concentrations of MNNG. This enzyme is also sensitive to inactivation by N-ethylmaleimide and mercurial reagents. The free sulfhydryl content of native enol-lactone hydrolase was determined to be two moles free sulfhydryl per mole of enzyme. A 95% inactivation of enol-lactone hydrolase by MNNG results in a masking of slightly more than one mole sulfhydryl per mole enzyme.Muconate lactonizing enzyme is moderately sensitive to inactivation by low concentrations of MNNG, but is not inactivated by sulfhydryl reagents. Muconolactone isomerase is resistant to inactivation by low concentrations of MNNG and is not inactivated by sulfhydryl reagents. Upon exposure to high concentrations of MNNG, muconolactone isomerase is rapidly inactivated. Spectrophotometric evidence indicates the lysine residues are nitroguanidinated proportionally with a loss in the enzymatic activity.These data indicate that the exposure of cells to low concentrations of MNNG should affect the activity of enzymes with essential sulfhydryl groups.     

Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B.

An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.