Seminal Plasma Fractions Research Articles

Characterization of proteins from boar prostate.

Most components of seminal plasma are secreted by accessory sexual glands: seminal vesicle, prostate gland and bulbourethral gland. The portion of proteins secreted by prostate gland differs in various species. Characterization of boar prostate proteins is the subject of this communication. Proteins of boar prostate gland were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H-) and heparin-binding (H+) fractions. The H- and H+ fractions were subjected to reverse phase high performance liquid chromatography (RP HPLC) and their elution profiles were compared with those of the H- and H+ fractions of boar seminal plasma. The isolated proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunodetection, N-terminal amino acid sequencing and mass spectrometry (MALDI). The following proteins of boar prostate secretion were identified: beta-microseminoprotein, serotransferrin, serum albumin, myoglobin and PSP I and PSP II spermadhesins. Presented results demonstrate composition of the main proteins of boar prostate secretion. Beta-Microseminoprotein was found to be a major protein of prostate secretion. PSP I and PSP II, major proteins of the H- fraction of boar seminal plasma, were found in boar prostate secretion in lower amounts. The major proteins of the H+ fraction of boar seminal plasma (AQN, AWN) were not detected in prostate secretion.

Detection and characterization of hepatitis C virus RNA in seminal plasma and spermatozoon fractions of semen from patients attempting medically assisted conception.

To investigate the risk of transmission of hepatitis C virus (HCV) via semen in assisted reproduction techniques, semen samples from 32 men chronically infected with HCV attending a center for assisted procreation were tested for HCV RNA by a reverse transcription-PCR protocol by using a modified version of the Cobas AMPLICOR HCV assay (version 2.0; Roche Diagnostics). The sensitivity of the test was 40 copies/ml. Four of 32 seminal plasma samples (12.5%) were found to be positive for the presence of HCV RNA. The median HCV load in blood was significantly higher in patients who were found to be positive for the presence of HCV RNA in semen than in those who tested negative (P = 0.02). In one man, seven consecutive seminal plasma samples tested positive for HCV RNA, as did two consecutive motile spermatozoon fractions; the corresponding fractions obtained after migration of the spermatozoa remained negative. Despite the absence of the proven infectivity of virus in semen samples that test positive for HCV RNA, these findings highlight the fact that seminal fluid may exhibit prolonged HCV RNA excretion. The usefulness of HCV RNA detection in both seminal plasma and spermatozoon fractions before the start of a program of medically assisted reproduction in couples in whom the male partner is chronically infected with HCV would need to be evaluated prospectively with a larger population of subjects exhibiting HCV RNA in their semen.

Mutual interactions of boar seminal plasma proteins studied by immunological and chromatographic methods

Mammalian fertilization includes highly regulated biochemical interactions: binding of seminal plasma proteins to the sperm surface during ejaculation, interaction of sperm surface proteins with oviductal epithelial cells, sperm capacitation, gamete recognition, primary binding of the sperm to the ovum, etc. Spermadhesins, sperm binding proteins of seminal plasma, play roles in all these events. The plasma membrane of spermatozoa undergoes extensive remodeling when sperm mixes with seminal plasma proteins at ejaculation. Some proteins of seminal plasma become sperm‐coating agents. Two types of interactions could be involved in this process: (i) the interaction of proteins with the sperm membrane, (ii) mutual interactions between protein components. The second type of interactions is the subject of the present communication. Proteins of boar seminal plasma have been shown to be present mostly as aggregates of different composition and binding properties. Mutual interactions of proteins of boar seminal plasma likely to form aggregates were investigated by gel chromatography and affinity chromatography on immobilized spermadhesins. The protein composition of boar seminal plasma fractions adsorbed to immobilized spermadhesins was found to correspond to that of aggregated forms of seminal plasma proteins. Mutual interactions of isolated proteins were tested by gel chromatography and by specific antibodies against spermadhesins. The results obtained confirmed the existence of complex formation between spermadhesins of the AQN and AWN families, as well as between proteins involved in the formation of heterodimer PSP I/PSP II. Aggregated forms of spermadhesins are likely to be physiologically important in fertilization.

Pharmacokinetic Profiles of Nevirapine and Indinavir in Various Fractions of Seminal Plasma

Pharmacokinetic Profiles of Nevirapine and Indinavir in Various Fractions of Seminal Plasma

Surface of human sperm bears three differently charged CD52 forms, two of which remain stably bound to sperm after capacitation.

gp20 is a sialoglycoprotein of the human sperm surface with a core peptide homologous to the leukocyte antigen CD52, a GPI-anchored glycosylated protein which is described by the monoclonal antibody CAMPATH-1. Comparative analyses, by means of CAMPATH and anti-gp20, indicated that they describe it in morphologically and functionally different ways, suggesting that the respective epitopes are different but also casting doubt on the immunological identity of the antigen. In the present study, we used immunodepletion to demonstrate that CAMPATH and anti-gp20 interact with the same antigen, but that anti-gp20 has a much higher avidity for the antigen than CAMPATH. Anion exchange fractionation analysis of the antigen revealed three differently charged gp20-CD52 forms, the least charged of which, was largely without a GPI-anchor. All three forms were associated with freshly ejaculated sperm, whereas capacitated sperm only contained the two GPI-anchored, more charged forms, which were also the ones found in the prostasome fraction of seminal plasma and in leukocytes. The two charged, GPI-anchored forms were described as homogeneous by anti-gp20, since they ran as a singlet; the third form ran as a doublet. When tested for insertion into Jurkat T cells, the medium charged form inserted the most readily and the less charged one could not be inserted at all.

A low-molecular-weight fraction of human seminal plasma activates adenylyl cyclase and induces caspase 3-independent apoptosis in prostatic epithelial cells by decreasing mitochondrial potential and Bcl-2/Bax ratio.

The majority of elderly men are affected by benign and malign diseases of the prostate that are governed by endocrine factors and local stromal/epithelial and luminal/epithelial interactions. Prostate epithelial cells secrete numerous factors into the seminal plasma (SMP) that are thought to be responsible for nutrition, accurate pH, and ionic environment of sperm. Our hypothesis assumes that prostatic factors responsible for optimal fertility might have retrograde influences on epithelial cell growth, differentiation, and function. SMP was analyzed for proteins and other biologically active substances by size exclusion high-performance liquid chromatography. Each fraction was investigated for its effect on cell growth and death. A low molecular mass fraction (2-4 kDa) was responsible for inducing apoptosis in proliferating prostate epithelial cells. Signal transduction was mediated by the production of cAMP; no significant changes in tyrosine phosphorylation of membrane receptors were observed. Mechanisms of apoptosis, i.e., caspase- and mitochondria-dependent pathways, were investigated in prostate epithelial cells by caspase activity assays, annexin/propidium iodide staining, changes in mitochondrial potential, p53, Par-4, Bax, and Bcl-2 protein levels. SMP induced p53- and Bcl-2-dependent apoptosis without activation of caspase-3. Obviously, SMP contains protective factors that help eliminate degenerated cells and control epithelial renewal. Age-related changes in the composition of SMP or the susceptibility of epithelial cells might, therefore, contribute to proliferative prostatic diseases

Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers

Objective: To measure folate levels in seminal plasma from smokers and nonsmokers and to evaluate relationships between seminal plasma folate levels and both folate nutriture and semen quality measures. Design: Observational study. Setting: United States Department of Agriculture, Western Human Nutrition Research Center, Presidio of San Francisco, San Francisco, California. Patient(s): Healthy male smokers (n=24) and nonsmokers (n=24). Main Outcome Measure(s): Blood levels of plasma folate and homocysteine, seminal plasma total, non-methyl- and 5-methyltetrahydrofolate concentrations, and total sperm count and density. Results: Total seminal plasma folate concentrations were on average 1.5 times higher than blood plasma folate concentrations in all men. Seminal plasma folates contained 5-methyltetrahyrdofolate (74% of total) and non-methyltetrahydrofolates (26% of total); all samples had less than four glutamyl residues. Total and 5-methyltetrahydrofolate concentrations correlated significantly with blood plasma folate and homocysteine concentrations. Seminal plasma non-methyltetrahydrofolate levels correlated significantly with sperm density and total sperm count. Seminal plasma of smokers contained a proportionally lower concentration of non-methyltetrahydrofolates compared with nonsmokers. Conclusion(s): Seminal plasma total folate and 5-methyltetrahydrofolate concentrations reflect folate nutriture. The non-methyltetrahydrofolate fraction of seminal plasma may be important for male reproductive function.

Inhibition of the antibody responses to rat blood transfusion antigens in mice by boar seminal immunosuppressive fraction.

Immunosuppressive fraction of boar seminal vesicle fluid (ISF) was tested to muffle primary and secondary antibody responses to xenotranfusions. Contemporaneously, heparin non-binding fraction of seminal plasma (H- fraction), presumed to be identical to ISF, was used to support the results. To study their similarity, ISF and H- fraction were analyzed by high-performance liquid chromatography and the separated proteins by N-terminal sequencing. In sera of mice treated with ISF or H- fraction, the productions of antibodies against rat erythrocytes and blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The productions of IgM, IgA, and IgG subclasses were followed by sandwich ELISA. ISF and H- fraction were proved to be equal complexes of porcine seminal plasma (PSP) proteins PSP I and PSP II. Both inhibited antibody responses to rat erythrocytes and serum and the concentrations of IgM, IgG, IgG1 and IgG2 after the first transfusion with a long-lasting effect. Both suppressed the secondary antibody production if applied before the second transfusion. IgA and IgG3 stayed uninfluenced. ISF and H- fraction had an equal immunosuppressive effect. ISF was characterized biochemically, found to be identical to H- fraction, and determined to be powerful in overcoming unwanted exaggerated antibody responses to xenotransfusion.

Inhibition of acrosine-like protease activity by a lectin affinity chromatographic bovine seminal plasma fraction containing the PDC-109 and aSFP proteins

These studies showed that the fractionation of bovine seminal plasma based on lectin agarose affinity chromatography, employing lectins specific to asparagine linked oligosaccharides, and a lectin specific for fucosylated glycans, lead to products with an inhibitory effect on the acrosine-like protease activity. This effect decreases when glycocompounds containing fucosylated Lewis x structures are removed, suggesting that these compounds might have some role in the modulation of this activity in the bull. In the fraction devoid of high mannose, hybrid and non-bisecting lactosaminic oligosaccharide-containing glycocompounds, PDC-109 and aSFP proteins were detected and characterized at microscale.

Distribution of lipid and protein in human semen fractions

Human semen is formed by the secretions of different glands. We fractionated semen by centrifugation and obtained four main fractions: (a) spermatozoa, (b) material precipitating at 10 000× g, (c) prostasomes (precipitate at 105 000× g), and (d) a soluble fraction. When required, fractions were purified further. We find that most semen protein (about 85%) is in the soluble fraction, 7% in spermatozoa and the remainder is scattered in the other fractions. We compared the electrophoretic pattern of soluble protein with the protein of prostasomes and found marked differences. On the other hand, prostasomes, that comprises only about 3% of total semen protein, contain about 45% of cholesterol and almost 15% of lipid phosphorus with a cholesterol to phospholipid molar ratio greater than 2. On the contrary, phospholipid is largely bound to the fraction containing spermatozoa (about 46% of total lipid phosphorus). This fraction is poor in cholesterol and has a cholesterol to phospholipid molar ratio of about 0.2. The distribution of lipid phosphorus among lipid classes shows some similarity in the soluble fraction and in prostasomes; in both fractions, sphingomyelin is the most abundant phospholipid (about 50%). On the other hand, phosphatidylcholine is the main phospholipid in spermatozoa-enriched fractions (about 35% of total lipid phosphorus). We conclude that the various fractions of seminal plasma obtained by centrifugation differ markedly from each other as to lipid and protein content.

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.

Detection and quantification of HIV-1 in semen: identification of a subpopulation of men at high potential risk of viral sexual transmission.

To assess HIV burden in both acellular and cellular fractions of semen in men with different levels of blood plasma HIV RNA by a cross-sectional study. Fifty-two HIV-1-seropositive men (21 receiving antiretroviral therapy) with CD4 cell counts ranging from 1 to 1170 x 10(6)/l. Semen was separated into seminal plasma and fractions enriched in motile spermatozoa or non-spermatozoal cells. HIV RNA was quantified by the HIV-Monitor technique (Roche) in blood plasma, seminal plasma and spermatozoa fractions. HIV DNA or infectious virions in cellular fractions were detected by either PCR or qualitative viral culture. HIV RNA was detected in 86.5% of seminal plasma specimens and in 14.6% of spermatozoa fractions; HIV DNA was detected in 57.1% of non-spermatozoal cell fractions. HIV RNA levels in blood plasma and seminal plasma were correlated (r5 = 0.56, P < 0.0001, Spearman's rank test). A majority of men had lower levels in seminal plasma than in blood plasma: one-third had HIV-positive seminal cell fractions. However, 20 men (38.5%) with HIV RNA levels in seminal plasma (median: 4.65 log10 copies/ml) comparable to or higher than those in blood plasma had all HIV-positive non-spermatozoal cells or spermatozoa fractions with a high frequency of positive cultures. A high frequency of men had detectable HIV in semen. We identified a subpopulation demonstrating high levels of HIV RNA in seminal plasma, comparable to or higher than those in blood plasma, frequently associated with a substantial viral shedding in seminal cells, raising the possibility of viral production within the genital tract and suggesting heterogeneity in the potential of HIV sexual transmission among infected men.

Seminal plasma components stimulate interleukin-8 and interleukin-10 release.

Human seminal plasma has potent anti-inflammatory properties which are thought to confer a survival advantage to the spermatozoa within the hostile female genital tract. In contrast, a profound pro-inflammatory leukocytosis has been observed post-coitus in animals and humans. Whether components of seminal plasma are involved in initiating this leukocytic reaction is not known. This study investigated the effect of human seminal plasma, a seminal plasma fraction and its principal constituent prostaglandins, prostaglandin E2 (PGE2) and 19-hydroxy PGE, on the release of the pro-inflammatory neutrophil chemotactic factor interleukin-8 (IL-8) and the anti-inflammatory cytokines interleukin-10 (IL-10) and secretory leukocyte protease inhibitor (SLPI). The tissues studied were non-pregnant cervical explants, peripheral blood and the monocyte cell line U937. Seminal plasma fraction (SPF) significantly (P < 0.05) stimulated release of IL-8 and inhibited release of SLPI from non-pregnant cervical explants. SPF, PGE2 and 19-hydroxy PGE significantly (P< 0.005) stimulated IL-8 release from peripheral blood and U937 cells. 19-hydroxy PGE was significantly (P< 0.005) more effective than PGE2 in stimulating IL-8 release. Seminal plasma, SPF and PGE2 significantly (P < 0.05) stimulated IL-10 release from U937 cells. 19-hydroxy PGE stimulated IL-10 release from U937 cells but this failed to reach significance. Release of IL-10 by cervical explants and SLPI by peripheral blood and U937 cells were below the detection limit of the assays employed. We suggest that the anti- and pro-inflammatory immune responses which seminal plasma induces might act in combination initially to promote sperm survival and then to facilitate their removal from the female genital tract.

Suppression of PMN-chemotaxis by different molecular weight fractions of equine seminal plasma

Suppression of PMN-chemotaxis by different molecular weight fractions of equine seminal plasma

Detection of HIV-1 in seminal plasma and seminal cells of HIV-1 seropositive men

It is of critical importance to precisely understand the modalities of HIV presence in semen, especially with regard to procreation. In this study, paired blood and semen samples from 31 human immunodeficiency virus type 1 (HIV-1)-positive men were assessed for cell-free HIV-RNA load in blood plasma (BP) and seminal plasma (SP), and for detection of HIV by culture, PCR and RT-PCR in semen cellular fractions separated by centrifugation on Percoll gradient. HIV-RNA was detected in 94% of BP and 84% of SP samples. For 11 men (35%), HIV-RNA load in SP was equal or superior to that observed in blood. HIV-DNA presence was demonstrated (either by PCR or culture positivity) in 39% of the non-spermatozoal cells (NSC)-enriched fractions, and in one Percoll-selected sperm pellet. HIV-RNA was detected in 17% (4/23) of the sperm pellets. This positivity was associated with an HIV-RNA load in SP equal or superior to the HIV-RNA load in blood, a high rate of HIV-DNA detection in the NSC fraction, and a low CD4+ cell count. In such conditions, a significant viral production inside the genital tract is more likely to be present, and a close association between HIV and gametes might occur. Assisted procreation with selected spermatozoa should be preceded by accurate assessments of viral presence in blood, SP and semen cellular fractions.

Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma.

Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.

Identification of Porcine Reproductive and Respiratory Syndrome Virus in Semen and Tissues from Vasectomized and Nonvasectomized Boars

Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV) can be identified in and transmitted through boar semen. However, the site(s) of replication indicating the origin of PRRSV in semen has not been identified. To determine how PRRSV enters boar semen, five vasectomized and two nonvasectomized PRRSV-seronegative boars were intranasally inoculated with PRRSV isolate VR-2332. Semen was collected three times weekly from each boar and separated into cellular and cell-free (seminal plasma) fractions. Both fractions were evaluated by reverse transcriptase nested polymerase chain reaction (RT-nPCR) for the presence of PRRSV RNA. Viremia and serostatus were evaluated once weekly, and boars were euthanatized 21 days postinoculation (DPI). Tissues were collected and evaluated by RT-nPCR, virus isolation (VI), and immunohistochemistry to identify PRRSV RNA, infectious virus, or viral antigen, respectively. PRRSV RNA was identified in semen from all vasectomized and nonvasectomized boars and was most consistently found in the cell fraction, within cells identified with a macrophage marker. Viral replication as determined by VI was predominately found within lymphoid tissue. However, PRRSV RNA was widely disseminated throughout many tissues, including the reproductive tract at 21 DPI. These results indicate that PRRSV can enter semen independent of testicular or epididymal tissues, and the source of PRRSV in semen is virus-infected monocytes/macrophages or non-cell-associated virus in serum. PRRSV-infected macrophages in semen may result from infection of local tissue macrophages or may originate from PRRSV-infected circulating monocytes or macrophages.

Improvement of cryopreserved ram sperm heterogeneity and viability by addition of seminal plasma.

The effect of seminal plasma, as well as some seminal plasma fractions, on the heterogeneity and viability of frozen-thawed ram spermatozoa was studied. Fresh and frozen sperm samples were simultaneously assessed by a routine semen-quality assay and centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system analysis. All samples used for this study were obtained from Salz rams by an artificial vagina and frozen according to the Fiser method. Sperm samples, consisting of either raw or washed semen, were frozen in the presence of either whole ram seminal plasma (RSP) or > 10 kDa seminal plasma fraction. Two control samples were considered: one diluted in Fiser's extender and an undiluted sample. Cell-membrane integrity (viability), hypoosmotic swelling (HOS) test, and sperm motility were assessed in all cases after collection, before freezing (at 5 degrees C), after thawing, and after removing the diluent before CCCD analysis. Our results show that sperm cell partition behavior was dependent on the sperm treatment and that the freezing-thawing process accounted for a clear loss of sperm heterogeneity and membrane integrity. These losses were less apparent when seminal plasma had been removed from semen samples before freezing. In addition, washed semen frozen in the presence of either > 10 kDa seminal plasma fraction or whole re-added seminal plasma maintained a higher rate of sperm heterogeneity and viability.

Prostasome fraction of human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist progesterone.

Seminal plasma prevents human sperm from becoming acrosomally responsive. These experiments tested the idea that the inhibitory activity of seminal plasma is contained in the particulate prostasome fraction. Most of the inhibitory activity was sedimentable (105,000 g, 2 h) and the majority of the recovered activity was in the prostasome fraction. The recovery of inhibitory activity in the prostasome fraction (42% of the activity in unfractionated seminal plasma) was similar to the recovery of cholesterol in that fraction (41%), consistent with cholesterol's role as the major inhibitor in seminal plasma. To test whether components of the prostasome fraction bind to sperm, the prostasome fraction was made fluorescent with fluorescein isothiocyanate or with the acetoxymethyl ester of 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. No labeled material was seen to bind to sperm, suggesting that exchange of cholesterol between prostasomes and sperm takes place through the aqueous phase or at the time of vesicle-sperm collision.

The Isolation from Human Seminal Plasma of a New Form of Soluble 5′-Nucleotidase

Soluble broad spectrum 5′-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min−1mg−1. SDS–PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+and Mn2+were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Piwas a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[α,β-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.