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.     

Ouabain- and potassium-induced stimulation of amylase release in fragments and acini of rabbit pancreas

Ouabain increases the enzyme secretion from the isolated rabbit pancreas and pancreatic fragments, but not from isolated pancreatic acini. The increase occurs after a delay of 45-60 min and is not accompanied by an increase in lactate dehydrogenase release. The stimulatory effect of ouabain (10(-5) M) is dependent on the presence of extracellular calcium, and is not antagonized by 10(-4) M atropin, 10(-4) M propranolol, 10(-5) M phentolamine, 10(-3) M dibutyryl-cyclic GMP, 10(-6) M tetrodotoxin, 10(-4) M verapamil or 10(-4) M D-600. Elevation of the extracellular potassium concentration to 120 mM in the presence of 10(-4) M atropin also increases the enzyme secretion from rabbit pancreatic fragments. The increase is again dependent on the presence of extracellular calcium and is resistant to adrenergic blockade and to tetrodotoxin, verapamil or D-600. Forskolin also stimulates a Ca2+-dependent release of amylase from pancreatic fragments but not from pancreatic acini. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IMX), ouabain (10(-5) M) and K+ (120 mM) cause an immediate increase in the cyclic AMP content of pancreatic fragments which does not occur in the absence of extracellular calcium. In pancreatic acini, the cAMP production is only slightly increased by ouabain. In the absence of IMX, the cAMP levels in fragments or acini are not detectably altered by ouabain or K+. The results suggest that the stimulation of enzyme secretion by ouabain and high K+ is an indirect effect, mediated by the release of an endogenous transmitter from non-cholinergic, non-adrenergic nerves in the intact preparations. The release and/or the effect of the transmitter appears to be mediated primarily by Ca2+ and secondarily by cyclic AMP.     

Fluidity characteristics of bovine thyroid plasma membranes

Highly purified plasma membranes of bovine thyroid were obtained by differential pelleting followed by discontinuous gradient centrifugation in a swing-out rotor. Subfractions of plasma membranes were prepared by affinity chromatography on Con A-Sepharose. The final membrane fractions were enriched 25-30-fold over homogenate in 5'-nucleotidase and alkaline phosphatase and displayed a protein to phospholipid ratio of 1.67 and a cholesterol to phospholipid molar ratio of 0.55. The phospholipid composition did not deviate appreciably from that of whole tissue except for the higher sphingomyelin level (22.5 vs. 14.0%). The predominant fatty acids were palmitic (16:0), oleic (18:1), stearic (18:0) and linoleic (18:2) acid. The physical state of the membrane was studied by (i) calculation of the lipid structural order parameter SDPH from steady-state fluorescence anisotropy determinations of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH); (ii) estimation of the lateral diffusion coefficient of pyrene following excimer formation. These parameters were determined in native thyroid plasma membranes and in reconstituted vesicles, obtained by detergent dialysis from octylglucoside solubilized membrane components. The presence of membrane protein or neutral lipids induced more restraint on the movements of the fluorophores. The lipid order parameter, SDPH was mainly determined by the neutral lipids. Subfractions of plasma membrane enriched in luminal membranes have a slightly lower fluidity (higher SDPH and lower Ddiff values) than subfractions enriched in basolateral membranes. This difference appears to be due to both differences in lipid as well as protein composition. Under physiological conditions, no significant alterations in probe dynamics could be observed upon addition of thyrotropin or cholera toxin, even at micromolar concentrations.     

Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach

To determine the yield of radiation-induced single-strand, double-strand and potential breaks (breaks which are converted into actual breaks by alkali or heat treatment) oxygenated aqueous solutions of phi X174 supercoiled circular double-stranded (RFI) DNA were irradiated with increasing doses of gamma-irradiation and subjected to electrophoresis on agarose gels both before and after heat treatment. A complete separation was obtained of RFI, RFII (relaxed circle due to one or more single-strand breaks) and RFIII (linear DNA due to one double-strand break). A computer-assisted spectrophotometric procedure was developed, which enabled us to measure very accurately the amount of DNA present in the three DNA fractions. The quantitative changes of each fraction of DNA with dose could be fitted to a straightforward statistical model, which described the dose-dependent formation of the different types of breaks and from which the D37-values of single-strand, potential single-strand and double-strand breaks could be calculated to be 0.42 +/- 0.02, 1.40 +/- 0.25 and 57 +/- 36 Gy respectively. Potential double-strand breaks were not formed significantly under our conditions. In addition the maximum distance between two independently introduced single-strand breaks in opposite strands resulting in a double-strand break could be determined. The values before and after heat treatment are shown to be 29 +/- 6 and 102 +/- 13 nucleotides, respectively.     

Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol

Rat reticulocytes were incubated with rat 125I-Tf-59Fe under conditions inhibiting heme synthesis. Cytosol, prepared from the reticulocytes, was separated and analysed by gel filtration and Amicon Ultrafiltration. An iron-containing low molecular weight fraction derived from the cytosol was further analysed by HPLC size-exclusion chromatography and HPLC reversed phase chromatography. Conditions inhibiting heme synthesis and uncoupling the oxidative phosphorylations lead to a large increase in the Fe-containing low molecular weight fraction in the cytosol. The components in the low molecular weight fraction have an apparent molecular weight of 5500 Dalton as determined with HPLC size-exclusion chromatography. The low molecular weight fraction contained several iron chelating components like glycin, 1/2 cystine and citrate, but no specific iron-binding proteins, nucleotides or pyrophosphate.     

Investigation by pyrolysis mass spectrometry,phage pattern and plasmid analysis of staphylococci that have reverted from ‘L’-phase to bacterial phase

No major differences have been found in series of Staphylococcus aureus strains which reverted from L-phase, either by pyrolysis mass spectrometry or by phage-typing or sensitivity testing. In L-phase they have been subcultured for a long time or transformed/reverted many times into/from L-phase. Plasmids were lost during transformations/reversions, but there was some difference between the tetracycline-connected plasmids on the one hand and the erythromycin-connected ones on the other.This investigation was supported by the Foundation for Fundamental Research on Matter (F.O.M.), subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants [Phaseolus vulgaris (L.) Savi.]

This study was initiated to characterize Rhizobium isolates obtained from root nodules of ineffectively nodulated, field-grown alfalfa (Medicago sativa L.) plants. The purpose was to determine if these isolates possessed characteristics which would explain either their ineffectiveness in N₂ fixation or their apparent ability to tolerate the moderately acid soil conditions from which they originated. Isolates were characterized by analysis of growth rate, 39°C tolerance, acid production on conventional media, and symbiotic performance. All isolates were ineffective in N₂ fixation on alfalfa, and they contained one or more anomalous characteristics. These included either slow growth rate, lack of 39°C tolerance, or lack of acid production on conventional media. Infectiveness tests on a broad range of legumes revealed that the isolates formed root nodules on M. sativa, Medicago lupulina L., and Phaseolus vulgaris (L.) Savi. (common bean). These results provide evidence that, in some situations, ineffective nodulation of M. sativa in the field may be due to the presence of promiscuous, native Rhizobium species.