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Embryos and larvae of sea urchins (Lytechinus variegatus, Strongylocentrotus droebachiensis, Strongylocentrotus purpuratus, Dendraster excentricus), and starfish (Pisaster ochraceus) were investigated for the presence of a functional endocannabinoid system. Anandamide (arachidonoyl ethanolamide, AEA), was measured in early L. variegatus embryos by liquid chromatography/mass spectrometry. AEA showed a strong developmental dynamic, increasing more than 5-fold between the 8-16 cell and mid-blastula 2 stage. 'Perturb-and-rescue' experiments in different sea urchin species and starfish showed that AEA blocked transition of embryos from the blastula to the gastrula stage, but had no effect on cleavage divisions, even at high doses. The non-selective cannabinoid receptor agonist, CP55940, had similar effects, but unlike AEA, also blocked cleavage divisions. CB1 antagonists, AEA transport inhibitors, and the cation channel transient membrane potential receptor V1 (TrpV1) agonist, arachidonoyl vanillic acid (arvanil), as well as arachidonoyl serotonin and dopamine (AA-5-HT, AA-DA) acted as rescue substances, partially or totally preventing abnormal embryonic phenotypes elicited by AEA or CP55940. Radioligand binding of [(3)H]CP55940 to membrane preparations from embryos/larvae failed to show significant binding, consistent with the lack of CB receptor orthologs in the sea urchin genome. However, when binding was conducted on whole cell lysates, a small amount of [(3)H]CP55940 binding was observed at the pluteus stage that was displaced by the CB2 antagonist, SR144528. Since AEA is known to bind with high affinity to TrpV1 and to certain G-protein-coupled receptors (GPCRs), the ability of arvanil, AA-5-HT and AA-DA to rescue embryos from AEA teratogenesis suggests that in sea urchins AEA and other endocannabinoids may utilize both Trp and GPCR orthologs. This possibility was explored using bioinformatic and phylogenetic tools to identify candidate orthologs in the S. purpuratus sea urchin genome. Candidate TrpA1 and TrpV1 orthologs were identified. The TrpA1 ortholog fell within a monophyletic clade, including both vertebrate and invertebrate orthologs, whereas the TrpV1 orthologs fell within two distinct TrpV-like invertebrate clades. One of the sea urchin TrpV orthologs was more closely related to the vertebrate epithelial calcium channels (TrpV5-6 family) than to the vertebrate TrpV1-4 family, as determined using profile-hidden Markov model (HMM) searches. Candidate dopamine and adrenergic GPCR orthologs were identified in the sea urchin genome, but no cannabinoid GPCRs were found, consistent with earlier studies. Candidate dopamine D(1), D(2) or alpha(1)-adrenergic receptor orthologs were identified as potential progenitors to the vertebrate cannabinoid receptors using HMM searches, depending on whether the multiple sequence alignment of CB receptor sequences consisted only of urochordate and cephalochordate sequences or also included vertebrate sequences., (Copyright 2009 S. Karger AG, Basel.)

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.

N-Acylethanolamines (NAEs) are an important family of lipid-signaling molecules. Arachidonylethanolamide (anandamide) (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) are co-produced from similar phospholipid precursors when neurons are stimulated. AEA is an endogenous agonist (endocannabinoid) for cannabinoid receptors. It binds with higher affinity to type CB1 than to type CB2 cannabinoid receptors. PEA does not bind to CB1, while the hypothesis that it reacts with putative CB2-like receptors has been questioned. OEA does not activate currently known cannabinoid receptors, but it mimics the effects of AEA and cannabinoids in reducing the fertilizing capacity of sea urchin sperm. OEA and PEA also act as entourage compounds by inhibiting the hydrolysis of AEA by fatty acid amide hydrolase. Cannabinoid receptors and/or AEA are present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, preimplantation embryo and placenta, as well as prostatic and mammary carcinomas. We now report that analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) shows the presence of AEA, PEA, and OEA in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk, and fluids from malignant ovarian cysts. Previous studies showed that AEA-signaling via cannabinoid receptors regulates capacitation and fertilizing potential of human sperm, early embryonic development and blastocyst implantation into the uterine mucosa of rodents, as well as proliferation of human mammary and prostatic carcinomas. Current results imply that NAEs also may modulate follicular maturation and ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior. Collectively, these findings suggest that NAEs in human reproductive fluids may help regulate multiple physiological and pathological processes in the reproductive system, and imply that exogenous cannabinoids delivered by marijuana smoke might impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs. Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors. We now report that AEA is present in human seminal plasma, mid-cycle oviductal fluid, and follicular fluid analyzed by high-performance liquid chromatography/mass spectrometry. Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. Specific binding of the potent cannabinoid agonist [(3)H]CP-55,940 to human sperm was saturable (K(D) 9.71 +/- 1.04 nM), suggesting that they express cannabinoid receptors. R-methanandamide [AM-356], a potent and metabolically stable AEA analog, and (-)delta(9) tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro. AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. Both AM-356 and THC inhibited morphological alterations over acrosomal caps between 2 and 6 hr (IC(50) 5.9 +/- 0.6 pM and 3.5 +/- 1.5 nM, respectively). Sperm fertilizing capacity, measured in the Hemizona Assay, was reduced 50% by (1 nM) AM-356. These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts, and imply that smoking of marijuana could impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids., (Copyright 2002 Wiley-Liss, Inc.)

Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation.

Delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of the sperm. Sperm fertility depends upon their motility, and their capacity to undergo the acrosome reaction upon encountering a specific ligand derived from the egg's jelly coat. The acrosome reaction involves exocytosis of the acrosomal granule at the apex of the sperm head and elongation of the acrosomal filament. This process exposes the sperm membrane that will attach to and fuse with the egg. Pretreatment of sperm with THC prevents the triggering of the acrosome reaction by solubilized egg jelly in a dose and time dependent manner. Motility of THC-treated sperm is not reduced compared to control sperm in sea water or vehicle dissolved in sea water. The adverse effects of THC on the acrosome reaction and sperm-fertilizing capacity are reversible. Studies with ionophores suggest that THC blocks the acrosome reaction by affecting event(s) in the stimulation-secretion coupling mechanism in the sperm preceding the opening of ion channels. Ultrastructural studies show that THC, CBD and CBN block the membrane fusion reaction between the sperm's plasma membrane and the acrosomal membrane that normally is elicited in response to stimulation by egg jelly to initiate the acrosome reaction. However, lipid deposits are found in the subacrosomal and centriolar fossae of cannabinoid treated sperm. The nuclear envelope is fragmented in close proximity to the lipid deposits within the subacrosomal fossa. These morphological observations suggest that cannabinoids may activate phospholipase(s) within the sperm. Biochemical studies show that THC activates phospholipase A2 activity in sperm homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the egg jelly induced acrosome reaction by delta 9-tetrahydrocannabinol (THC) is associated with the localized disruption of the nuclear envelope and the formation of lipid deposits in sea urchin sperm. This suggests that THC may activate phospholipase(s) within the sperm. We now report effects of THC on phospholipase A2 activity in homogenates of sea urchin sperm using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine as substrate. The release of radioactive arachidonic acid was measured after a 30-min incubation with the enzyme. In the absence of exogenous Ca2+, 100 microM THC produced a significant (P less than 0.001) increase in phospholipase A2 activity. THC activated phospholipase A2 in a concentration (1-100 microM) and time-dependent (0-30 min) manner. Exogenous calcium (10 mM) significantly augmented basal (P less than 0.001) and THC-stimulated (P less than 0.005) phospholipase A2 activity. Calcium chelators [ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] inhibited the basal level of phospholipase A2 activity in the sperm homogenate, and prevented the activation of phospholipase A2 by THC. Submicromolar levels of free calcium ions were required for THC stimulation of phospholipase A2. Cannabinol which mimics the effects of THC on the acrosome reaction also activated phospholipase A2 in sperm homogenate. These results suggest that THC may alter lipid metabolism in sperm by activating calcium-dependent phospholipase A2. Putative metabolites derived from this process may inhibit the acrosome reaction and thereby reduce the fertilizing capacity of sea urchin sperm.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.

Cannabinoids are potent pharmacological substances derived from marihuana. The effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) on fertilization in the sea urchin Strongylocentrotus purpuratus were investigated. Insemination of THC-treated eggs (5-400 microM) with excess sperm did not result in polyspermic fertilization. At minimal sperm densities, THC (0.1-10 microM) inhibited fertilization in a dose-dependent manner. Pretreatment of eggs with THC did not reduce their receptivity to sperm. Pretreatment of sperm with THC reduced their fertilizing capacity. The concentration of THC required to reduce sperm fertility by 50% was 1.1 +/- 1.1 microM. The fertilizing capacity of THC-treated sperm depended on concentration of sperm and duration of pretreatment. The fertility of sperm at minimal densities was reduced by 50% at 129.3 +/- 43 s treatment with 10 microM THC. The adverse effect of THC on sperm fertility was reversible. CBN and CBD at comparable concentrations (0.1-10 microM) inhibited fertilization in a manner similar to THC. First division was not delayed in zygotes that were fertilized with sperm pretreated with 10 microM THC. These studies show that cannabinoids directly affect the process of fertilization in sea urchins by reducing the fertilizing capacity of sperm.

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, alpha-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymotrypsin."

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.

Sea urchin sperm contain a phenylhydrazine-sensitive peroxidase that is believed to use hydrogen peroxide produced by the fertilized egg to reduce sperm fertility and thereby assist in the prevention of polyspermy. Strongylocentrotus purpuratus sperm were treated initially with hypotonic phosphate buffer (pH 7.0) to remove catalase and then extracted with 0.5% Triton X-100 in 0.5 M acetate buffer (pH 5.0). Peroxidase activity in this detergent extract was assayed using 3,3',5,5'-tetramethyl benzidine (TMB) as oxidizable substrate. Kinetic studies showed that the Km for TMB is 250 microM. Benzohydroxamic acid and phenylhydrazine are known to be competitive inhibitors of a variety of plant and animal peroxidases. These substances were found to competitively inhibit the sea urchin sperm peroxidase: for benzohydroxamic acid, Ki = 51.2 microM, mean inhibitory dose (ID50) = 146.7 microM; for phenylhydrazine, Ki = 201 nM, ID50 = 303 nM. These findings indicate that the biochemical properties of the sea urchin sperm peroxidase resembles those of peroxidases found in somatic tissues where oxygen radicals are produced by phagocytes to kill bacteria and support our hypothesis that the sperm peroxidase has a functional role in the prevention of polyspermy during fertilization.

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.