Ethanolamine base exchange enzymatic activities in spontaneous transformed glial cell lines. Effect of dibutyryl cyclic AMP treatment

Cultured astrocytes derived from neonatal rats (normal cells) displayed maximal ethanolamine base exchange enzymatic activity (EBEE) when cultures reached confluency and cells almost ceased to divide. At this stage, ethanolamine phosphotransferase (EPT) and choline base exchange enzyme (CBEE) activities reached a plateau. In spontaneously transformed glial cells, no differential activity variation either between EPT and CBEE, or between EPT and EBEE was observed. The EBEE activity was mainly localized in the microsomal fraction and was completely absent from plasma membranes. Dibutyryl cyclic AMP (db-cAMP) treatment of the transformed cells reversed the pattern of these activities to that of normal cells. Moreover, treatment of the transformed cells with medium conditioned by normal astroblasts markedly increased EBEE activity. This study demonstrates that (i) variation of EBEE activity during cell growth differs in normal and in transformed cultured glial cells. (ii) EBEE activity may be modulated via both db-cAMP and normal cell conditioned medium. Our findings suggest a possible implication of EBEE in the maturation and contact inhibition of cell growth.     

Ethanolamine base exchange in astrocyte primary cultures: Localization and developmental studies

The enzymatic activities of ethanolamine base exchange (EBEE) and CDP-ethanolamine: 1,2-diacylglycerol ethanolamine phosphotransferase (EPT) were investigated during the growth of rat astrocyte primary cultures. From the 16th day, cells ceased to divide (2.0×10⁶ cells per culture dish); the total phospholipid (PL) content increased 1.5 fold between the 16th and 24th day (0.20 to 0.30 mol per mg protein) but the amount of ethanolamine phospholipid (28% of PL content) remained constant. Whereas the specific activity (pmol/ min × mg protein) of EPT reached a plateau at 16 days in culture and remained constant (400) thereafter, that of EBEE increased up to the 19th day (190) and decreased gradually to a basal level (75) at the 24th day. EBEE activity was not detected in plasma membranes isolated from 16, 19 and 24 days astrocyte cultures. Sub-cellular fractionation and determination of EBEE specific activities showed that (1) the 104×10³ g fraction (P4) was 4.8 and 8.8 fold enriched at the 16th day and 24th day respectively as compared to the whole cell homogenate (50 and 75). (2) the 7×10³ g (P2) and 17×10³ g (P3) fractions were 8.4 and 7.0 fold enriched respectively at the 19 day in culture. The percentages of the enzymatic activity in the different subcellular fractions were 30, 57.2 and 25.7 for P2 and 39.2, 2.6 and 39.8 for P4 at 16, 19 and 24 days in culture respectively. The activity remained constant in P3 (23%) and was negligible in P1 (6%). Ultrastructual studies revealed that P2 and P3 were enriched in mitochondria while P4 contained essentially microsomes. P4 was enriched in glucose-6-phosphatase activity (G-6-P microsomal marker) and P2 and P3, in monoaminooxidase (MAO) and succinate dehydrogenase (SDH) (mitochondrial markers); G-6-P, MAO and SDH in the different subcellular fractions remained constant from the 16th to the 24th day. These data indicate (1) that the rate and profile of EPT and EBEE activities differed during the differentiation of astrocyte culture; (2) that EBEE activity, except at the 19th day in culture, was mainly localized in a microsomal subcellular fraction; (3) that at the 19th day the optimal EBEE activity observed in whole cell homogenate correlates with an enrichment of this activity in an enriched mitochondrial subcellular fraction.     

Phospholipid base exchange in human leukocyte membranes: quantitation and correlation with other phospholipid biosynthetic pathways

The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca2+ concentration, but markedly inhibited by Mg2+. Despite this Ca2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05-0.2 mg/ml; Con A, 0.5-2 micrograms/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N-transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A2 activity.     

Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital

Hepatic triglyceride lipase (HTGL) was measured in primary rat hepatocytes maintained for 3 days under three different culture conditions: basal medium, basal medium plus insulin, and basal medium plus insulin and phenobarbital. The activity of HTGL secreted by these cells was measured by treating intact cells with heparin; intracellular enzyme was subsequently measured in cell homogenates. Insulin stimulated intracellular triglyceride lipase activity by 48% and extracellular lipase by 30%. Phenobarbital, an enzyme-inducing drug, caused a further 15% increase in extracellular hepatic triglyceride lipase; whereas, the intracellular activity was reduced. The presence of insulin greatly stimulated the rate of enzyme secretion, and this rate was not notably affected by the presence of phenobarbital. After 3 days in culture, the short term (2-8 h) synthesis and secretion of enzyme from cultures treated with insulin or insulin plus phenobarbital were equally inhibited by cycloheximide. Monensin also inhibited enzyme secretion in both cultures and caused a similar increase in intracellular lipase activities. Insulin did not significantly affect the proportion of intracellular enzyme (17.7% basal vs. 15.8% insulin). On the other hand phenobarbital produced a 20-30% reduction in the proportion of intracellular enzyme (12.5 vs. 17.7% basal or 15.8% insulin). These findings suggest a drug-induced redistribution of triglyceride lipase.     

Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

The Effects of Various Incubation Temperatures, Particulate Isolation, and Possible Role of Calmodulin on the Activity of the Base Exchange Enzymes of Rat Brain

The involvement of calmodulin in the choline, ethanolamine, and serine exchange activities of rat brain microsomes was investigated. Calmodulin stimulated choline exchange activity to a greater extent than ethanolamine and serine exchange activities. The three base exchange activities were inhibited by antipsychotic drugs believed to prevent calmodulin interaction, but not by calmodulin-binding protein. The solutions employed for tissue homogenization and subsequent isolation of microsomes greatly influenced the base exchange activities. The process of resuspending isolated microsomes and recentrifugation, or "washing," produced major losses of detectable activity. The base exchange enzyme activities were maximal at 45 degrees, and Arrhenius plots revealed a common transition temperature of 31 degrees. The activation energies for the base exchange reactions decreased at temperatures above the observed transition temperature. Kinetic data, Km and Vmax, for the base exchange activities at 27, 37, and 45 degrees are presented.     

Phosphatidylethanolamine synthesis by castor bean endosperm : a base exchange reaction

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl(2). l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

Separate myocardial ethanolamine phosphotransferase activities responsible for plasmenylethanolamine and phosphatidylethanolamine synthesis

Ethanolamine phosphotransferase (EPT) is a key enzyme responsible for the synthesis of ethanolamine glycerophospholipids. Plasmenylethanolamine is a predominant molecular subclass of ethanolamine glycerophospholipids in the heart. The present study was designed to identify the selective use of 1-O-alk-1'-enyl-2-acyl-sn-glycerol as a substrate for EPT as a mechanism responsible for the predominance of plasmenylethanolamine in the rabbit heart. EPT activity in rabbit myocardial membranes using 1,2-diacyl-sn-glycerol as substrate is activated by Mn2+, inhibited by dithiobisnitrobenzoic acid (DTNB) and is unaffected by Ca2+. In contrast, ethanolamine phosphotransferase activity using 1-O-alk-1'-enyl-2-acyl-sn-glycerol as substrate is inhibited by Mn2+ and Ca2+, but is activated by DTNB. Additionally, ethanolamine phosphotransferase activity using 1-O-alk-1'-enyl-2-acyl-sn-glycerol substrate was more sensitive to thermal denaturation compared with that of 1,2-diacyl-sn-glycerol. Taken together, these results suggest that separate ethanolamine phosphotransferase activities are present in heart membranes that are responsible for the synthesis of phosphatidylethanolamine and plasmenylethanolamine.     

Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions

Abstract: Enrichment in the base-exchange activities was found in the micro-somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin-sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin-insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane-bound base exchange activities. Some activity was restored in phospholipase C-treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A-treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D-treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.     

Activation of ornithine decarboxylase in monolayer cells treated with trypsin

When monolayer Chinese hamster cells are treated with trypsin for short periods of time, ornithine decarboxylase (ODCase) activity increases two- to fourfold. This increase can be blocked by aprotinin, a protease inhibitor, and is not observed when cultures are dislodged from substrate mechanically prior to contact with exogenous trypsin. The trypsin-induced increase in ornithine decarboxylase activity is not due to degradation of enzyme or inhibitor molecules or to new enzyme synthesis. Immunoprecipitable protein, radiolabeled with [3H]alpha-difluoromethylornithine in vitro, is the same molecular weight in cells harvested with or without trypsin. Protein-bound levels of this specific enzyme-activated irreversible inhibitor of ornithine decarboxylase are unchanged by trypsin treatments that increase enzyme activity. Trypsin treatment of rat embryonic fibroblasts, transformed by a temperature-sensitive mutant of Rous sarcoma virus, increases ODCase activity in cells growing at the nonpermissive, but not at the permissive, temperature for the transformed phenotype. These results suggest that ornithine decarboxylase can be activated by exogenous trypsin treatment in a manner that is dependent on cell adhesion properties, which are modified in transformed cells.     

Sphingosine and unsaturated fatty acids modulate the base exchange enzyme activities of rat brain membranes

The base exchange enzymes catalyze the incorporation of L-serine, ethanolamine and choline into their corresponding phospholipids. The L-serine base enzyme activity was increased 120% by 0.1 mM sphingosine. There was a modest increase of the ethanolamine base exchange enzyme activity but the choline base exchange enzyme activity was unaffected. Na-arachadonate, Na-oleate and Na-linolenate at 0.2 mM concentration increased the activity of the L-serine and ethanolamine base exchange enzymes but inhibited the choline base exchange enzyme activity. A model is proposed suggesting that modulations of the L-serine base exchange enzyme may participate in the regulation of the calcium phospholipid-dependent protein kinase C.     

Dexamethasone increases the incorporation of [3H] serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: A possible relation between serine base exchange enzyme and apoptosis

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [³H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.     

Properties of a murine monocytic tumor cell line J-774 in vitro. II. Enzyme activities.

Lysosomal enzyme activities, collagen degrading activity and sensitivity to bacterial infection were tested in a murine monocytic cell line, J-774, during cultivation with or without fetal calf serum (FCS) or endotoxin, and compared with the same parameters in normal murine peritoneal macrophages. The basic intracellular level of two out of three lysosomal enzyme activities tested (acid phosphatase and β-glucuronidase) and their extracellular release were higher in the J-774 cells than in normal macrophages, indicating that the tumor cells were more “activated”. This was further supported by the moderate increase in intracellular enzyme activities after FCS and endotoxin stimulation of the J-774 cells. Normal macrophages showed a much more impressive rise in these parameters after stimulation. Collagen-degrading activity was found at the same magnitude, or lower, in tumor cell cultures, compared to normal macrophage cultures. However, the activity in the tumor cultures was enhanced by endotoxin stimulation. The J-774 cells showed a higher sensitivity to bacterial contamination, tested after E. coli addition to the cultures, than normal macrophages. This high sensitivity could be prevented by pretreatment of the tumor cells with endotoxin.     

Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells.

A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.     

Phosphatidylethanolamine synthesis by castor bean endosperm : membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl₂. l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model

Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.     

Plasminogen activator from human embryonic kidney cell cultures: Evidence for a proactivator

The nature of the trypsin-activatable plasminogen activator produced by kidney cell cultures (Bernik, M.B. (1973), J. Clin. Invest. 52, 823–834) was investigated using human embryonic kidney (HEK) cell cultures in serum-free medium. Plaminogen activator activity ratios (trypsin-activated/ untreated controls) in HEK cell-conditioned media were maximal (up to 3) during the first week of culture and remained nearly constant at approximately 2 for the next 3–5 weeks, while the total plasminogen activator titer increased in a nearly linear manner. Therefore, coincident with progressive cell dengeration and death, the ratios decreased to near unity due to “spontaneous” activation of the enzyme, which was inhibited in cell-free conditioned media by the pancreatic trypsin inhibitor Kunitz and benzamidine. Since the activator is not inhibited by the trypsin inhibitor, it is concluded that a protease other than the plasminogen activator is responsible for the activation. Increases in the plasminogen activator titers (about 2-fold) were similarly obtained by culturing the cells in medium containing low concentrations (0.05–0.10 μg/ml) of trypsin for up to about 6 weeks. The presence of the trypsin inhibitor in HEK cell cultures decreased the rate of activation, resulting in higher activity ratios (up to 6), and the total plasminogen activator activity was reduced only minimally (<20%), if at all, by the highest concentration of the trypsin inhibitor (100 μg/ml) tested.Affinity chromatography of conditioned media with activity ratios of 1.6–2 separated the plasminogen activator into an active fraction and a fraction which was activated a minimum of 200-fold by trypsin and contained no measurable activity prior to activation. Gel filtration of crude conditioned media or partially purified activator separated the plasminogen activator activity into two peaks; both were trypsin-activatable, and their relative proportions varied with the isolation conditions. The results indicate the occurrence of a proenzyme form of the plasminogen activator in the culture media.     

The histochemistry of developing adipocytes in primary stromal-vascular cultures of rat adipose tissue

Summary Histochemical analysis for NADP-dependent dehydrogenases, succinate dehydrogenase, NADH and NADPH-tetrazoleum reductases and esterase was conducted on primary cultures of adipose tissue stromal-vascular cells. Enzyme activities were restricted to clusters of lipid laden cells (adipocytes). The number of enzyme reactive adipocytes increased with length of culture. Coverslips were partially coated with collagen to allow comparisons of cell differentiation on coated (C-glass) and uncoated glass (U-glass) surface. There were no reactions for NADH- and NADPH-tetrazoleum reductases (TR) in cells on C-glass whereas adipocytes and stromal cells on U-glass were reactive. Glucose-6-phosphate (G6PDH) and 6-phosphogluconate (6PGDH) dehydrogenase activities were markedly demonstrated in both stromal cells and adipocytes on U-glass. Malate (MDH) and isocitrate (ICDH) dehydrogenase activites were higher in adipocytes than in stromal cells on the U-glass. Stromal cells on C-glass were either devoid of these enzymes (G6PDH, MDH, 6PGDH, ICDH) or activity was restricted to a small area of the cytoplasm. There were two levels of staining intensity in (MDH, ICDH, G6PDH, 6PGDH) adipocyte clusters on C-glass.Elimination of phenazine methosulphate from the NADP-dependent dehydrogeanse medias and SDH media, caused a reduction in enzyme reactive adipocytes on the C-glass. This manipulation did not reduce the number of enzyme reactive cells on U-glass. Cells on C-glass and U-glass were distinctly different in esterase stained coverslips. These studies demonstrated enzyme histochemical reactions of adipocytes and stromal cells in primary culture that were dependent on the type of extracellular matrix. Furthermore, enzyme histochemistry was shown to be useful for delineating adipocytes from stromal cells in primary cultures.     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Enchancement of streptococcal transformation yield by proteolytic enzymes.

Trypsin and other proteolytic enzymes, added together with transforming DNA or during cell-DNA contact to competent cultures of several streptococcal strains, enchanced (10 to 600%) the yield of genetic transformation (stimulation). With few exceptions, the level of stimulation was high (over 100%) when competence was low (below 2%). Stimulation was caused by the action of an enzyme on competent cells and not on any other component of transformation mixture. The phenomenon occurred when the enzyme was added to the culture not earlier than 7 min before and not later than 5 min after the period of cell-DNA contact. The presence of trypsin during cell-DNA contact caused: (i) the alterations at cell surface, demonstrated by electron microscopy, increased release of 3H-amino acid-labeled material, and higher cell susceptibility to autolysis; (ii) the increase of both total and irreversible binding of DNA by the cells; and (iii) the decrease of early nucleolytic degradation of DNA by cells. These and other data point to the importance of a delicate balance of recipient cell's surface nuclease activities in the effectiveness of transformation process. It is also possible that trypsin eliminates an unknown cellular factor which obstructs DNA-cell receptors interaction.