Phosphoprotein phosphate activity at the outer surface of intact normal and transformed 3T3 fibroblasts

Using ³²P-labeled phosphocasein or phosphohistones as exogenous substrates it was possible to detect a phosphoprotein phosphate activity on the outer surface of intact normal and transformed 3T3 fibroblasts. Incubation of monolayers of intact cells in buffered salt solution with the radioactively labeled substrate resulted in the release of alkali-labile ³²P counts into the surrounding medium. The reaction was: (a) linear with time (at least up to 20 min); (b) proportional to the cell density; (c) dependent on the temperature and pH of the incubation medium; (d) stimulated by K⁺; and (e) inhibited by sodium fluoride, inorganic pyrophosphate, zinc chloride and relatively impermeant sulfhydryl reagents. Less than 2% of the externally located phosphoprotein phosphatase activity was detectable in pooled cell-free washings of the intact cell monolayer. Phosphocasein did not cause any detectable leakage of intracellular lactate dehydrogenase or soluble phosphoprotein phosphatase activity into the external medium; incubation of the cells with phosphohistones, on the other hand, resulted in appreaciable leakage of both these cytoplasmic activities. Neoplastic transformation was associated with a nearly two-fold decrease in the activity of the surface phosphoprotein phosphatase. Addition of serum to either non-transformed 3T3 or spontaneously transformed 3T6 cells resulted in a rapid and remarkable drop in the cell surface dephosphorylating activity. Acrylamide gel electrophoresis of the dephosphorylated casein or histone substrate revealed no proteolytic degradation or change in electrophoretic mobility. The intact cells showed no damage upon microscopic examination as a result of exposure to phosphocasein or phosphohistones.     

Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system.     

Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation.

Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.     

Occurrence of an ecto-phosphoprotein phosphatase in goat epididymal spermatozoa

Intact spermatozoa from goat cauda epididymis possess phosphoprotein phosphatase activity that causes dephosphorylation of externally added [32p]histones. The enzymic reaction was linear with time for at least 15 min and there was little uptake of [32p]histones by these cells. The activity of the enzyme of the whole spermatozoa was not due to contamination of the broken cells or epididymal plasma and leakage of the intracellular enzymic activity during incubation. The activity of the phosphoprotein phosphatase was strongly inhibited by the thiol reagent: p-chloromercuribenzenesulfonic acid, which is believed not to enter the cells. There was no appreciable loss of the enzymic activity from the cells when washed with EDTA (2.0 mM) or a hyperosmotic medium. These data are consistent with the view that the observed activity of the enzyme is located on the spermatozoal external surface. Studies with unlabelled p-nitrophenyl phosphate and beta-glycerophosphate indicate that the sperm ecto-enzyme is not a non-specific phosphatase.     

Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties

The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.     

Passive membrane permeability to small molecules and ions in transformed mammalian cells: probable role of surface phosphorylation

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.     

Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.     

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.     

Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.     

A specific effect of external ATP on the permeability of transformed 3T3 cells

Addition of ATP causes a dramatic increase in the rate of p-nitrophenyl phosphate hydrolysis by intact 3T6 and 3T3 cells transformed by polyoma virus and simian virus 40 (SV40). In sharp contrast, untransformed 3T3 cells or secondary mouse embryo fibroblasts, either growing or resting, do not respond to ATP. The activation displays specificity, reversibility and dependence on pH, temperature and ATP concentration. The data suggest that exposure to ATP changes the permeability of transformed cells to p-nitrophenyl phosphate thus revealing an internal, ouabain-insensitive, phosphatase activity.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Secretary sialidase activity and GM3 ganglioside

The sialidase activities with GM3 ganglioside and sialyllactitol were demonstrated in the conditioned medium of human fibroblasts. pH versus activity profiles of conditioned medium with GM3 as substrate suggested the presence of two sialidases with optimal activities at pH 4.5 and pH 6.5. The GM3 sialidase activity at pH 6.5 was suppressed in the medium of contact-inhibited cells. This sialidase may function in the metabolism of cell surface GM3 since there was a selective loss of labeled sialic acid from GM3 at different times of incubation after pulse-labeling with a radioactive sialic acid precursor ([3H]N-acetyl-mannosamine) and a radioactive ceramide precursor ([14C]serine). In addition, a sialidase inhibitor, 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (NeuAc-2-en) resulted in a reversible growth inhibitory effect and the suppression of the sialidase activity in the medium. We have speculated that GM3 hydrolysis on the cell surface by the sialidase may be coordinated with the cell cycle and may be at its maximum during early in the G1 phase.     

Purification and properties of a phosphoprotein phosphatase from rat liver.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Effect of cultivation conditions on the growth properties of Swiss 3T3 cells

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells

Summary Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium -glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.