Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?     

Electrophoretic abnormalities of lysosomal enzymes in mucolipidosis fibroblast lines.

Electrophoretic properties of eight lysosomal hydrolases and 36 nonlysosomal enzymes were investigated in cultured fibroblasts from children with the inherited storage disease mucolipidosis II (ML II); fibroblasts from a child with a related disorder, mucolipidosis III (ML III); and two obligate heterozygous cell lines from parents of a ML II child. Cell homogenates of ML II fibroblast lines showed altered mobilities for lysosomal beta-hexosaminidase, acid phosphatase2, and alpha-mannosidase and deficient activity for the esterase-A4 and lysosomal alpha-mannosidase-B electrophoretic phenotypes. Altered mobility was also detected for the nonlysosomal enzyme adenosine deaminase-d. Deficient activities of other lysosomal enzymes were observed as previously reported. In a single ML III fibroblast line, only beta-hexosaminidase showed an abnormal electrophoretic pattern suggesting a difference between these cells and ML II fibroblasts. Thirty-five nonlysosomal enzymes associated with other cellular organelles and metabolic pathways were electrophoretically normal in all mucolipidosis cell lines. Heterozygous ML II cells showed normal expression for all enzymes. Two major patterns of altered lysosomal enzymes and adenosine deaminase were demonstrated in ML II cell lines, suggesting that at least two genetic forms of this disorder may exist. Neuraminidase treatment of ML II homogenates converted altered forms of acid phosphatase2 and adenosine deaminase-d and in two ML II lines, recovered the previously undetected lysosomal alpha-mannosidase band. These results are consistent with the mucolipidosis defect(s) being associated with abnormal post-translatinal processing of multiple lysosomal enzymes and adenosine deaminase-d.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.     

Control of lysosomal acid phosphatase expression in man-mouse cell hybrids

Lysosomal acid phosphatase activity in human and mouse cells was separated into multiple zones by starch gel electrophoresis. One of the two major zones in the mouse was apparently extinguished when genetic information from man and the mouse was combined in proliferating man-mouse somatic cell hybrids. The evidence suggested that the absence of the mouse lysosomal acid phosphatase (mAP-1) was influenced by the human genome. The gene coding for human acid phosphatase (hAP-1) was shown to be unlinked to the presumed human component which extinguished the mouse acid phosphatase (mAP-1). The mechanism of “extinction” is postulated to be a modification in the processing of the mouse lysosomal enzyme. A dimeric structure was suggested for acid phosphatase-1 of man, mouse, and rat since a single hybrid enzyme was expressed in man-mouse and mouse-rat somatic cell hybrids.     

Partial Separation and Characterization of Soluble Phosphatases from Leaves of Wheat Grown Under Phosphorus Deficiency and Water Deficit

Soluble phosphatases from wheat leaves have been separated intotwo fractions by CM-cellulose chromatography. The phosphatase(s)in the fraction adsorbed by CM-cellulose (Fraction B) were identicalwith an electrophoretic band of phosphatase activity which increasedwith phosphorus (P) deficiency. The phosphatase activity inthis fraction increased specifically as the concentration ofphosphate in the sampled tissues fell from 0.8% to 0.2% dryweight. Phosphatase activity in Fraction B may be due to anisozyme of acid phosphatase. Phosphatase activity in the fractionnot adsorbed by CM-cellulose (Fraction A) increased with bothP deficiency and water deficit, and was higher in young thanin mature leaves. The Fraction A phosphatase which increasedwith water deficit was probably another isozyme of acid phosphatase:however the Fraction A phosphatase which increased with P deficiencywas probably not an acid phosphatase because it had activityonly against p-nitrophenylphosphate and ATP. In expanding leaves of P deficient plants, phosphatase activitiesin Fractions A and B declined after P was supplied to adequatelevels. However, in fully expanded leaves, high phosphataseactivities persisted after the resupply of P. These resultsare discussed in relation to the use of measurements of phosphataseactivity as a biochemical marker of P deficiency.     

Studies on the fractionation of mucosal homogenates from the small intestine

1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.     

Differential Expressions of Two Types of Alkaline Phosphatase during Developmental Growth of Embryoid Bodies of Mouse Teratocarcinoma OTT-6050 In Vitro

Ascites teratocarcinoma OTT-6050 is a totipotent tumor line producing indefinitely the simple type of embry-oid bodies (EBs). In culture with fetal calf serum (FCS) in Eagle's minimal essential medium (MEM), these EBs show developmental growth, only in which some differentiative events result. EBs also show this developmental growth in MEM supplemented with two fractions of FCS separated with a Amicon PM 10 membrane, i.e. a low molecular weight Fraction L (mol. wt. less than 10,000) and a high molecular weight Fraction H (mol. wt. more than 10,000). Fraction H is necessary for the survival of EBs in vitro. Fraction L enhances the uptake of ³H-thymidine into EB cells with increase in the Vmax , but no change in the K m. On culture of EBs with both Fractions, a marked bimodal increase in alkaline phosphatase (ALPase) activity is seen on day 1–2 and 4, resulting from the differential expressions of two electrophoretically distinct ALPases (Bands I and II). The differential expressions of ALPase are also observed cytochemically, one activity being on the inner cells and the other on the surrounding cells of EBs. From the cytochemical similarity of ALPase activity to that of normal mouse embryos, Band I ALPase is inferred to be the epiblast (developmentally totipotent stem cell)-type and Band II ALPase to be the distal (parietal) endoderm-type.     

Structural features of naegeli amylodextrin as indicated by enzymic degradation

Naegeli amylodextrin is the insoluble residue remaining after prolonged treatment of native starch granules with strong aqueous acid. The Naegeli amylodextrin from waxy maize starch was separated by gel chromatography on Sephadex G-50 into three fractions. Although the fractions were heterogeneous, their average structures were examined by enzymic degradation with porcine-pancreactic alpha amylase, beta-amylase, and pullulanase. The results show that Fraction I (highest molecular weight) has complex branching, Fraction II (major component, d.p. ～25) contains about one branch per molecule, and Fraction III (d.p. ～12) is mostly linear. Formation of these acid-resistant fractions may be explained as arising from a cluster model of amylopectin in which the outer chains are in a double-helical, crystalline arrangement.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell-by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6-phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme-cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology.     

Lysosomal tartrate sensitive acid phosphatase deficiency in cells which contain lysosomal "high uptake forms"

The catalytic and immunological properties of acid phosphatases (EC 3.1.3.2.) in different tissues were studied. It was demonstrated that high uptake forms of lysosomal enzymes like beta-galactosidase isolated from human platelets and bovine testis are mature enzymes, which have not lost their mannose-6phosphate marker. The results presented indicate that this phenomenon is related to a low activity or the complete absence of the lysosomal tartrate sensitive acid phosphatase activity in the tissues concerned.     

Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.

In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity.     

THE INFLUENCE OF THE MODE OF NUTRITION ON THE DIGESTIVE SYSTEM OF OCHROMONAS MALHAMENSIS

The intracellular distribution and level of acid hydrolases in Ochromonas malhamensis were studied in cells grown osmotrophically in a defined medium, in a carbon-free starvation medium, and during phagotrophy in each of these media. By cytochemical techniques, little enzymic reaction product was observed in the vacuoles of osmotrophic cells grown in the defined medium. Starved cells, however, contained autophagic vacuoles and cannibalized other Ochromonas cells. Dense enzymic reaction product was observed in the digestive vacuoles and in the Golgi cisternae of these starved cells. Moreover, starved cells and cells grown in a nutritionally complete medium ingested Escherichia coli which appeared in digestive vacuoles containing enzymic reaction product. Biochemical assays for lysosomal acid phosphatase (E.C. 3.1.3.2 orthophosphoric monoester phosphohydrolase) and acid ribonuclease (E.C. 2.7.7.16 ribonucleate nucleotido-2'-transferase) were done on Ochromonas cultures in the same experimental treatments and under identical assay conditions as the cytochemical study. During starvation, the acid hydrolase specific activities were consistently twice those found in cells grown in an osmotrophic complete medium. Ochromonas fed E. coli showed no increase in acid hydrolase specific activity as compared to controls not fed E. coli. The latency of lysosomal acid hydrolases in cells fixed with glutaraldehyde was reduced, suggesting that this fixative increases lysosomal membrane permeability and may release enzymes or their reaction products into the cytoplasmic matrix during cytochemical analysis. This could explain the cytoplasmic staining artifact sometimes observed with glutaraldehyde-fixed cells when studied by the Gomori technique. This study confirms that Ochromonas malhamensis, a phytoflagellate, does produce digestive vacuoles and can ingest bacteria, thereby fulfilling its role as a heterotroph in an aquatic food chain. When Ochromonas is grown in a nutritionally complete osmotrophic medium, phagocytosis causes appearance of acid hydrolases in the digestive vacuoles, whereas the total activity of the enzymes remains unchanged. An organic carbon-free medium strongly stimulates acid hydrolaes activity and causes these enzymes to appear in the digestive vacuoles whether phagocytosis occurs or not.     

Distribution of lysosomal enzymes in different types of rat liver cells.

Eight lysosomal enzymes were measured in different types of rat liver cells. Hepatocytes were purified by low speed centrifugation of a cell suspension obtained by treating the perfused liver with collagenase. Nonparenchymal cells (NPC) were purified by centrifugation after treating the initial cell suspension with pronase, which selectively destroys the parenchymal cells (PC). Kupffer cells were found to attach selectively to tissue culture dishes after overnight culture of an NPC suspension. The specific activity of lysosomal enzymes was generally higher in NPC than in hepatocytes, but the different enzymes were concentrated to different degrees in the NPC. Specific activity of acid phosphatase was 1.7 times higher in NPC than in hepatocytes. Specific activity of acid DNAase, on the other hand, was 8 times higher in NPC than in hepatocytes. Other enzymes showed intermediate values. Assuming that 30% of the liver cells are nonparenchymal it may be calculated that from 7% (acid phosphatase) to 25% (acid DNAase) of the hepatic lysosomal enzymes are present in the NPC. The pattern of lysosomal enzymes in cultured Kupffer cells was similar to that of the NPC from which the Kupffer cells were derived. Cathepsin D and β-glucuronidase were, however, elevated in Kupffer cells as compared with NPC. The enzyme pattern in Kupffer cells was almost identical with that of rat peritoneal macrophages.     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

Lysosomal Acid phosphatase deficiency: liver specific variant in the mouse

The number and classes of genes responsible for the final expression of lysosomal acid phosphatase were investigated in the mouse (Mus musculus ). In mouse tissues, lysosomal acid phosphatase activity was separated by gel electrophoresis into two major zones of activity. The cathodal zone of activity in liver of the SM/J inbred strain was almost completely absent, while the anodal zone was increased in activity. Other tissues from SM/J were not affected, nor were livers and other tissues in 27 inbred mouse strains. Genetic studies indicated that this deficiency variant segregated as an autosomal codominant gene which has been designated Apl to symbolize the acid phosphatase liver phenotype. The Apl gene was not linked to markers on chromosomes 1, 2, 4, 5, 7, 8, or X. Electrophoretic, heat denaturation, neuraminidase treatment, tartrate inhibition studies and tissue mixing experiments suggested that the Apl gene was not a structural gene for acid phosphatase, but a separate gene that functions in liver and is responsible for controlling or modifying an acid phosphatase structural gene product.     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish.

A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.