Development of a radioimmunoassay for colony stimulating factor.

Purified L-cell colony stimulating factor (CSF) and rabbit anti-CSF serum were used to devise a radioimmunoassay for this factor. The CSF was radiolabelled with the aid of lactoperoxidase and precipitated by a double antibody technique. Addition of unlabelled CSF caused a dose-related displacement of the labelled tracer. Similar results were noted with conditioned media and murine serum. The assay required only 4 days for completion as compared with 7 days for the conventional agar gel bioassay. Moreover, the radioimmunoassay proved more sensitive and accurate than the bioassay. This technique should allow further exploration of the role of CSF in granulopoiesis.     

High degree of homology between primary structure of human lysosomal acid phosphatase and human prostatic acid phosphatase

Alignment of the amino-acid sequences of the human lysosomal acid phosphatase (LAP) and human prostatic acid phosphatase (PAP) yielded an extensive homology between the two mature polypeptide chains. In the overlapping part, which extends over the entire PAP sequence and the N-terminal 90% of the LAP sequence, the identity is 49.1%. The LAP has an additional C-terminal sequence, which is encoded by the last exon of the LAP gene. This sequence contains the transmembrane domain of LAP, which is lacking in the secretory PAP. All six cysteine residues as well as 20 out of 27 (LAP) and 26 (PAP) proline residues present in the overlapping part of the proteins are conserved, suggesting that they are involved in stabilization of the tertiary structure of both proteins. Only two out of 8 N-glycosylation sites in LAP and 3 in PAP are conserved, suggesting that the dense N-glycosylation of LAP is related to its function in lysosomes.     

Mannose 6-phosphate/insulin-like growth factor II-binding proteins in human serum and urine. Their relation to the mannose 6-phosphate/insulin-like growth factor II receptor.

Human serum and urine contain polypeptides which bind mannose 6-phosphate (M6P) and insulin-like growth factor II (IGF II) and crossreact with antibodies against the M6P/IGF II receptor. These polypeptides are considered to be fragments of the M6P/IGF II receptor. The major Mr approx. 205,000 fragment in serum and urine is about 10 kDa smaller in size than the membrane-associated receptor and is accompanied by minor forms with Mr values ranging from 104,000 to 180,000. The presence of receptor fragments in biological fluids indicates that shedding is one of the mechanisms contributing to the turnover of the M6P/IGF II receptor and that receptor fragments are part of the heterogenous group of serum proteins whic bind IGF II.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Phosphorylation and sulfation of arylsulfatase A accompanies biosynthesis of the enzyme in normal and carcinoma cell lines

Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.     

Release from mouse macrophages of acidic isoferritins that suppress hematopoietic progenitor cells is induced by purified L cell colony stimulating factor and suppressed by human lactoferrin

Purified mouse L cell colony-stimulating factor (CSF) and purified iron-saturated human lactoferrin (LF) were assessed for their effects on release of acidic isoferritin-inhibitory activity (AIFIA) from resident peritoneal and spleen macrophages of B6D2F1 mice. Constitutive release of AIFIA was dependent on the number of macrophages conditioning the culture medium. Detection of release of AIFIA required at least 10(4) macrophages/ml, and increased release was noted with increased concentrations of cells. This release was enhanced by CSF and was induced by CSF from concentrations of 10(3) macrophages/ml, from which constitutive release of AIFIA was not detected. Increased concentrations of CSF induced increased release of AIFIA. The inducing effect was removed by pretreating CSF with rabbit anti-L cell CSF serum. LF suppressed the constitutive as well as the CSF-induced release of AIFIA, but results were dependent on the relative concentrations of LF and CSF used. The suppressive effects of LF were removed by pretreating LF with goat anti-human LF. Constitutive, but not CSF-induced, release of AIFIA could be ablated by removal of Ia antigen-positive macrophages with low concentrations of monoclonal anti-Ia plus complement. Treating macrophages with higher concentrations of anti-Ia in the absence of complement blocked the LF suppression of constitutive AIFIA release but not the CSF-induction of AIFIA release. Release of AIFIA from mouse macrophages can be modulated by CSF and LF. This modulation may be of significance for the regulation of myelopoiesis.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.     

The structural basis of anomalous kinetics of rabbit liver aryl sulfatase A

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K_i values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of ³⁵S-labeled substrate at pH values near the pH optimum (pH 5–6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.