Investigating on the Correlation Between Some Biological Activities of Marine Sponge-Associated Bacteria Extracts and Isolated Diketopiperazines.

Marine organisms have been considered as the richest sources of novel bioactive metabolites, which can be used for pharmaceutical purposes. In the last years, the interest for marine microorganisms has grown for their enormous biodiversity and for the evidence that many novel compounds isolated from marine invertebrates are really synthesized by their associated bacteria. Nevertheless, the discovery of a chemical communication Quorum sensing (QS) between bacterial cells and between bacteria and host has gained the researchers to expand the aim of their study toward the role of bacteria associated with marine invertebrates, such as marine sponge. In the present paper, we report the evaluation of biological activities of different extracts of bacteria Vibrio sp. and Bacillus sp. associated with marine sponges Dysidea avara and Ircinia variabilis, respectively. Moreover, we evaluated the biological activities of some diketopiperazines (DKPs), previously isolated, and able to activate QS mechanism. The results showed that all extracts, fractions, and DKPs showed low scavenging activity against DPPH and superoxide anion, low cytotoxic and anti-tyrosinase activities, but no antimicrobial and acetylcholinesterase inhibitory activities. One DKP [cyclo-(trans-4-hydroxy-L-prolyl-L-leucine)] has the highest α-glucosidase inhibitory activity even than the standard acarbose.

Mariniflexile aquimaris sp. nov., isolated from seawater, and emended description of the genus Mariniflexile Nedashkovskaya et al. 2006.

A Gram-staining-negative, non-flagellated, non-gliding and rod-shaped bacterial strain, designated HWR-17(T), was isolated from seawater of the Yellow Sea in Korea. Strain HWR-17(T) grew optimally at pH 7.0-8.0, at 30 °C and in the presence of 2% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain HWR-17(T) clustered with the two Mariniflexile species in the family Flavobacteriaceae, exhibiting 16S rRNA gene sequence similarity of 97.1-97.2% to their type strains and less than 95.7% sequence similarity to other members of the family Flavobacteriaceae. Strain HWR-17(T) contained MK-6 as the predominant menaquinone and iso-C(15:0) as the major fatty acid. The polar lipid profile of strain HWR-17(T) contained phosphatidylethanolamine, an unidentified aminolipid and four unidentified lipids. The DNA G+C content of strain HWR-17(T) was 35.7 mol% and it exhibited 11 and 10% DNA-DNA relatedness, respectively, with Mariniflexile gromovii KCTC 12570(T) and Mariniflexile fucanivorans DSM 18792(T). The phylogenetic and genetic distinctiveness and differential phenotypic properties revealed that strain HWR-17(T) is distinguishable from the two recognized Mariniflexile species. On the basis of the data presented, strain HWR-17(T) is considered to represent a novel species of the genus Mariniflexile, for which the name Mariniflexile aquimaris sp. nov. is proposed. The type strain is HWR-17(T) (=KCTC 23346(T) =CCUG 60529(T)). An emended description of the genus Mariniflexile is also proposed.

Hydro-alcoholic extract of Morus nigra reduces fasting blood glucose and HbA1c% in diabetic patients, probably via competitive and allosteric interaction with alpha-glucosidase enzyme; a clinical trial and in silico analysis.

OBJECTIVES: 1-Deoxynojirimycin (1-DNJ), the main active component found in Morus nigra (black mulberry) is reported to be effective in controlling diabetes. We have evaluated the effect of hydro-alcoholic extract of M. nigra leaves on the fasting blood glucose (FBS) and hemoglobin A1c% (HbA1c%) in diabetic patients. Furthermore, we compared the interaction of 1-DNJ and glucose molecules with the alpha-glucosidase enzyme, which has a critical role in the lysis of glucose-based polymers in human cells. METHODS: 4% hydro-alcoholic extract was prepared from black mulberry leaves. Patients in treatment (n=50) and control (n=50) groups received 3 mL extract or placebo in water, respectively, and three times a day. Fasting blood glucose and HbA1c% were evaluated before and after three months of evaluation. Potential binding sites of 1-DNJ or glucose on the enzyme glucosidase found by docking study. Docking scores were obtained using an energy minimization method by Molegro Virtual Docker software. The Mean ± SD of each variable was compared between groups at the 95% significant level. RESULTS: Age mean ± SD was equal to 54.79 ± 9.203 (38-69) years. There was no significant difference between intervention and placebo groups considering FBS (p=0.633) but was for HbA1c% (p=0.0011), before treatment. After three months, both FBS and HbA1c% were significantly reduced in patients under mulberry leaves extract-treatment. FBS changed was from 182.23 ± 38.65 to 161.23 ± 22.14 mg/dL in treatment group (p<0.001) and from 178.45 ± 39.46 to 166.23 ± 29.64 mg/dL in control group (p<0.001). HbA1c was changed from 7.23 ± 0.25 to 6.13 ± 0.61% in treatment group (p<0.001) and from 7.65 ± 0.85 to 7.12 ± 0.33% in control group (p=0.854). Docking results showed that 1-DNJ binds more efficiently, and with a significant score than glucose, to human alpha-glucosidase. CONCLUSIONS: This clinical trial and virtual analysis showed that a hydro-alcoholic extract of black mulberry (M. nigra) leaf may be efficient in reducing the blood glucose and HbA1c% in diabetic patients. Furthermore, docking studies propose a competitive and allosteric regulation for herbal ingredients. Drug-development could be based on the presented idea in this report.

Soil enzyme responses to land use change in the tropical rainforest of the Colombian Amazon region.

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.

Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes.

A method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of Acanthamoeba castellanii (Neff) that have been incubated with polystyrene latex beads. Cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. Identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays of acid phosphatase, alpha- and beta-glucosidase, and reduced nicotinamide adenine dinucleotide dehydrogenase. When phagocytosis was allowed to occur for longer periods the phagocytic vesicles appeared to fuse with each other and perhaps with digestive vacuoles. The resultant vesicles which contained many beads were heavier than those which consisted of only one bead or a few beads with a closely applied membrane. Ultrasonication ruptured the isolated vesicles, and the membranes could then be isolated in 30-50% yield based on phospholipid analysis. These membranes were essentially free of acid hydrolases and, presumably, other soluble proteins, as was also indicated by their low ratio of protein to phospholipid. The membranes have been prepared both as closed vesicles and as open sheets.

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II.

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.

Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border.

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = approximately 300,000) by SDS PAGE. To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization. When a detergent extract of [35S]methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of approximately 300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG. Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal anti-gp330 IgG failed to do so. When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences. When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule: maltase was concentrated (approximately 90%) on the microvilli, and gp330 was concentrated (approximately 90%) in the clathrin-coated apical invaginations located at the base of the microvilli. We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.

Resolution of granules from rabbit heterophil leukocytes into distinct populations by zonal sedimentation.

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of beta-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.

Tay-Sachs disease: generalized absence of a beta-D-N-acetylhexosaminidase component.

Two hexosaminidase components, separable by starch-gel electrophoresis and possessing both beta-D-N-acetylglucosaminidase and beta-D-N-acetylgalactosaminidase activity, are present in human tissues. One of these, hexosaminidase component A, is absent in brain, liver, kidney, skin, cultured skin fibroblasts, blood plasma, and leukocytes from nine patients with Tay-Sachs disease. Hexosaminidase assay may facilitate the early diagnosis of individuals homozygous for Tay-Sachs disease.

Significant activities of extracellular enzymes from a brown tide in the coastal waters of Qinhuangdao, China.

Brown tides of Aureococcus anophagefferens have occurred annually in the coastal waters of Qinhuangdao since 2009. High levels of dissolved organic matter (DOM) are always measured during bloom periods. Study focusing on the effect of DOM on the occurrences of brown tides in this area is scare by far. To analyze the efficiency of DOM hydrolysis by different groups of microorganisms and the possible influence of DOM on the formation of brown tides, extracellular enzymes such as α, ß-glucosidases (α, ß-GLUs), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) as well as other environmental parameters were analyzed during a pre-bloom period of A. anophagefferens in June 2014. Dissolved organic nitrogen (DON) and phosphorus (DOP) contributed more than half of the total dissolved nutrient pools. Approximately 60-70% of the enzyme activities were associated with phytoplankton of size >5 µm. The hydrolysis rates of LAP were approximately 5 to 20 fold higher than those of AP and α, ß-GLUs. The ratios of ß-GLU activities: LAP activities indicated the hydrolysis potential related to proteins rather than polysaccharides. The differences in turnover time among the enzymes suggested that DOP was firstly hydrolyzed and recycled in the water in the early minutes, followed by the hydrolysis of DON and dissolved organic carbon (DOC)(in hours). Results suggest that the hydrolysis of DOM, in particular DOP, might significantly contribute to the occurrences of brown tides in the coastal waters of Qinhuangdao, China.

Metabolism of circulating disaccharides in man and the rat.

The metabolism of circulating disaccharides was studied in adult humans and rats. After iv infusions of 10 g of either lactose, sucrose, or maltose in four adults, no rise in blood glucose was noted. A mean of 8.7+/-1.89 g of the lactose and 6.3+/-1.39 g of the sucrose was excreted in the 24-hour urine sample. Only 0.11+/-0.03 g of the infused maltose was recovered in the urine, suggesting that the maltose was metabolized.After injection of (14)C-labeled lactose and sucrose in rats, 6.2+/-2.7 and 7.6+/-2.4%, respectively, was oxidized to (14)CO(2) in 24 hours; 62.1+/-13.5 and 68.4+/-10.8% of the respective disaccharides was excreted into the urine. Conversely, after injection of (14)C-labeled maltose 54.6+/-7.0% was oxidized to (14)CO(2) and 4.8+/-3.9% excreted in the urine. The per cent of maltose oxidized to CO(2) was similar to that of glucose. In addition to small intestinal mucosa, homogenates of rat kidney, brain, and liver as well as serum were found to have measurable maltase activities. The role of these tissue maltases in the metabolism of circulating maltose and maltosyloligosaccharides is discussed.

Midgut protease activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

We assayed the relative activities of midgut proteolytic enzymes in individuals of the fourth (L(4)) and fifth (L(5)) instar of Apollo larvae, inhabiting Pieniny Mts (southern Poland). The comparisons between midgut tissue with glicocalyx (MT) and liquid midgut contents with peritrophic membrane (MC) were made. Optimal media pHs of the assayed proteolytic enzymes in P. apollo midgut samples were similar to those of other lepidopteran species. Endopeptidases, as well as carboxypeptidases, digested effectively in alkaline environment, while aminopeptidases were active in a broad pH range. Trypsin is probably the main endoprotease (correlation with caseinolytic activity in MC of L(5) larvae: r=0.606; p=0.004); however, its activity was low as compared with that in other leaf-eating Lepidoptera. This suggests a minor role of trypsin and chymotrypsin in protein digestion in Apollo larvae, probably due to limited availability of the leaf proteins. Instead, due to very high carboxypeptidase A activity in midgut tissue, the larvae obtain exogenous amino acids either directly or from oligopeptides and glycoproteins. High and significant positive correlations between the enzyme activity and glucosidase as well as galactosidase activities strongly support this opinion.

Leojaponic acids A and B, two new homologous terpenoids, isolated from Leonurus japonicus.

The present study aimed at isolation and purification of the bioactive terpenoids from the herb of Leonurus japonicus by chromatographic separations such as silica gel, sephadex LH-20 and C18 reversed phase silica gel, as well as preparative HPLC. As a result, leojaponic acids A (1, C17H24O4) and B (2, C18H26O4), two homologous terpenoids, together with (-)-loliolide (3), 1-(3-ethylphenyl) ethane-1, 2-diol (4) and dibutyl phthalate (5), were isolated from the EtOH extract of L. japonicus. All the chemical structures of the isolates were elucidated on the basis of 1D and 2D NMR analyses. Compounds 1 and 2 were new terpenoids, and Compounds 3 and 4 were isolated and identified for the first time from this plant. In addition, the α-glucosidase and tyrosinase inhibitory activity of the new compounds were evaluated.

The major yeast exoglucanase: an extracellular glycoprotein lacking the carbohydrate outer chain.

The major exoglucanase (1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) secreted by Saccharomyces cerevisiae contains protein, mannose and phosphate in a molar ratio of 1:27:1. When digested with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) it sequentially released two asparagine-linked oligosaccharide chains. Oligosaccharides were fractionated into a neutral and acidic component, each one accounting for 50% of the total carbohydrate. The neutral oligosaccharide consisted of a mixture of three homologues ranging from GlcNAc-(Man)12 to GlcNAc-(Man)14. The acidic carbohydrate was, in turn, split into two components. The major one (45% of the initial material) contained a phosphodiester bond and released only mannose when subjected to mild acid hydrolysis. From the filtration pattern, it was shown to be a mixture of oligosaccharides ranging from GlcNAc-(Man)11-P to GlcNAC-(Man)13-P. The minor phosphorylated component, which represented the residual carbohydrate (5%), contained a phosphomonoester bond. It was also heterogeneous in size, the several homologues having one mannose less than their counterparts from the phosphodiester oligosaccharide. These results clearly indicate that the addition of an outer chain of carbohydrate is not a requirement for the externalization of yeast glycoproteins.

The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications.

Chicken intestinal sucrase-isomaltase and maltase-glucoamylase have been isolated in their intact form by detergent solubilization and characterized as to their subunit composition and mode of anchoring in the brush-border membrane. Both are heterodimeric enzyme complexes composed of two subunits each of approximately 140 and 130 kDa. Contrary to the mammalian sucrase-isomaltase, chicken isomaltase was identified as the smaller of the two subunits. As was shown by hydrophobic labeling, only one of the two subunits in each heterodimer is anchored in the bilayer, the smaller 130 kDa isomaltase subunit of the sucrase-isomaltase complex, and the larger 140 kDa subunit of the maltase-glucoamylase complex. Both preparations contain a high-molecular weight polypeptide of approximately 250 kDa which in the case of sucrase-isomaltase could be identified by peptide mapping as a single-chain precursor not (yet) proteolytically processed to the final heterodimer. These first data on the mode of membrane anchoring of non-mammalian glycosidases indicate that they are synthesized, inserted into the membrane, and processed in ways similar to the mammalian enzymes. The fundamental unity between avian and mammalian sucrase-isomaltases suggests that the partial gene duplication of an ancestral isomaltase gene and the subsequent mutation of one of the active sites resulting in pro-sucrase-isomaltase has occurred prior to the separation of mammals from reptiles, i.e. more than 300 million years ago.

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.

Modifications of the molecular weight of membrane-bound nonspecific beta-glucosidase in type 1 Gaucher disease determined in situ by the radiation inactivation method.

The radiation inactivation method has been used to compare the molecular weight of the nonspecific membrane-bound beta-glucosidase in situ in normal human spleen and in that of two patients with Gaucher disease type 1. We report, in type 1 Gaucher spleen, the presence of a high molecular weight component (557 000) in addition to the normal low molecular weight component (97 800). The various possible hypotheses explaining this high molecular weight component are discussed.

Sugar hydrolases of the infant rat intestine and their arrangement of the brush border membrane.

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush bordermembrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physiocochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26--35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein. Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.

In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease.

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine.

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.