Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.     

GnRH stimulates LH release directly via inositol phosphate and indirectly via cAMP in African catfish

In African catfish, two gonadotropin-releasing hormone (GnRH) peptides have been identified: chicken GnRH (cGnRH)-II and catfish GnRH (cfGnRH). The GnRH receptors on pituitary cells producing gonadotropic hormone signal through inositol phosphate (IP) elevation followed by increases in intracellular calcium concentration (?Ca(2+)(i)). In primary pituitary cell cultures of male African catfish, both cGnRH-II and cfGnRH dose dependently elevated IP accumulation, ?Ca(2+)(i), and the release of the luteinizing hormone (LH)-like gonadotropin. In all cases, cGnRH-II was more potent than cfGnRH. The GnRH-stimulated LH release was not associated with elevated cAMP levels, and forskolin-induced cAMP elevation had no effect on LH release. With the use of pituitary tissue fragments, however, cAMP was elevated by GnRH, and forskolin was able to stimulate LH secretion. Incubating these fragments with antibodies against cfGnRH abolished the forskolin-induced LH release but did not compromise the forskolin-induced cAMP elevation. This suggests that cfGnRH-containing nerve terminals are present in pituitary tissue fragments and release cfGnRH via cAMP signaling on GnRH stimulation, whereas the GnRH receptors on gonadotrophs use IP/?Ca(2+)(i) to stimulate the release of LH.     

Inhibitory and stimulatory interactions between endogenous gonadotropin-releasing hormones in the African catfish (Clarias gariepinus)

In the brain of all vertebrate classes, chicken (c) GnRH-II ([His(5), Trp(7),Tyr(8)]GnRH, cGnRH-II) is expressed in the mesencephalon. In addition, at least one other form of GnRH is expressed in the preoptical area/hypothalamus. In the human pituitary stalk and the mouse median eminence, cGnRH-II is present together with mammalian GnRH. Similarly, in the pituitary of several teleost fish (e.g., goldfish and eel, but not salmon or trout), a teleost GnRH is found together with cGnRH-II. These GnRHs are not colocalized in the same cells. Hence, these GnRH peptides may differentially regulate gonadotropin secretion and, in addition, may exert their effects simultaneously. The current study therefore investigated the effects of combinations of the two forms of GnRH present in the African catfish (Clarias gariepinus) pituitary-cGnRH-II and catfish GnRH ([His(5),Asn(8)]GnRH, cfGnRH)-on the cytosolic free calcium concentration ([Ca(2+)](i)) in single, Fura-2-loaded catfish gonadotrophs, as well as their effects on both in vitro and in vivo LH secretion. Both inhibitory and stimulatory effects of combinations of cfGnRH and cGnRH-II on [Ca(2+)](i) were observed, which were mirrored by their effects on both in vitro and in vivo LH secretion. The following pattern became apparent. The effect of intermediate or maximal effective cfGnRH doses was inhibited by the simultaneous presence of subthreshold or borderline effective cGnRH-II doses. Conversely, subthreshold or borderline effective concentrations of cfGnRH enhanced the effects of intermediate and maximal concentrations of cGnRH-II. In addition, combinations of cfGnRH and cGnRH-II concentrations that were equally active when tested separately showed an additive effect. The observed interactions between the two GnRHs may be of particular physiological relevance in the control of seasonal LH levels in the African catfish, as well as in other teleost species. Moreover, the occurrence of mutual inhibitory and stimulatory interactions between endogenous GnRHs may be a widespread aspect of GnRH action in vertebrates.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function studies of five natural, including catfish and dogfish, gonadotropin-releasing hormones and eight analogs on reproduction in Thai catfish (Clarias macrocephalus).

Five distinct forms of gonadotropin-releasing hormone (GnRH) and their analogs, six of which are newly designed, were used to study reproduction in Thai catfish, Clarias macrocephalus. Determination was made for the percentage of fish that ovulated within 16-18 h; the percentage of eggs fertilized; and the percentage of larva that hatched and survived for 7 days. The results show, firstly, that natural chicken GnRH-II, which is identical with catfish GnRH-II, was significantly more effective at a dose of 300 micrograms/kg than the control injection for the induction of ovulation. Dogfish GnRH at the same dose was also significantly more effective than the control, but was not significantly different from chicken (catfish) GnRH-II for ovulation induction. The novel catfish GnRH-I, mammalian GnRH and salmon GnRH were not effective at 100, 150 or 300 micrograms/kg in Thai catfish. Secondly, 5 of 8 analogs of GnRH at a dose of 20 micrograms/kg resulted in a significantly higher percentage of ovulating fish compared with the control fish. Among these five analogs, the most effective were the two analog forms of chicken GnRH-II (D-Arg6,Pro9 NEt and D-Nal6,Pro9 NEt), followed by the salmon GnRH analog (D-Arg6,Pro9 NEt), a dogfish GnRH analog (D-Arg6,Pro9 NEt) and the mammalian GnRH analog (D-Ala6,Pro9 NEt). Not significantly different from the controls were the two catfish GnRH-I analogs and one of the dogfish (D-Nal6,Pro9 NEt) analogs. The six new analogs had not been previously tested in any animal. Thirdly, the number of fish ovulating was the same whether GnRH was administered in one or two injections.     

Conformational constraint of mammalian,chicken, and salmon GnRHs,but not GnRH II,enhances binding at mammalian and nonmammalian receptors: evidence for preconfiguration of GnRH II

Mammalian GnRH (mGnRH) is believed to interact with mGnRH type I receptors in a beta-II' turn conformation involving residues 5-8. This conformation can be constrained by substitution of a D-amino acid at position 6 or by a lactam ring involving residues 6 and 7, thereby increasing receptor binding affinity. It has been proposed that this is not the case for non-mGnRH receptors. However, we show that this conformational constraint increases the binding affinity of mammalian, chicken, and salmon GnRH for the chicken and catfish receptors, as well as for the mouse receptor. Therefore, we conclude that the beta-II' turn conformation enhances ligand binding for non-mGnRH as well as mGnRH type I receptors. In contrast, most substitutions of a D-amino acid in position 6 have limited effect on binding affinity for GnRH II. We suggest that this ligand is preconfigured through intramolecular interactions, which accounts for its high binding affinity and total conservation of primary structure over 500 million years of evolution.     

Gonadotropin-releasing hormone-immunoreactive neuronal structures in the brain and pituitary of the African catfish,Clarias gariepinus (Burchell)

Summary The distribution of gonadotropin-releasing hormone (GnRH) immunoreactivity was studied in the African catfish, Clarias gariepinus, by means of immunofluorescence and immunoperoxidase techniques. Immunoreactive neurons were found throughout the preoptic nucleus (NPO). However, only a portion of the secretory perikarya in the NPO showed a positive reaction by use of an anti-LHRH serum. Numerous immunoreactive fibres were found to enter the pituitary and to terminate in its proximal pars distalis, the site of concentration of the gonadotropic cells. Since GnRH is present in the brain and pituitary of the African catfish, the lack of spontaneous ovulation in captivity is apparently due to an insufficient release of GnRH.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.     

Gonadal steroids and the maturation of the species-specific gonadotropin-releasing hormone system in brain and pituitary of the male African catfish (Clarias gariepinus)

The effect of testosterone (T), 11-ketotestosterone (KT) and estradiol (E(2)) on the development of the catfish gonadotropin-releasing hormone system (cfGnRH) of male African catfish (Clarias gariepinus), at the onset of puberty [between 10 and 12 weeks post hatching (ph)] was investigated. The cfGnRH neurons, located in the ventral forebrain, were visualized by immunofluorescence and their numbers were determined and the amounts of cfGnRH-associated peptide (cfGAP) in the pituitary were measured by RIA. Steroid treatments did not significantly alter the numbers of immunoreactive GnRH neurons. However, T and E(2) caused an increase in the amount of GnRH, demonstrated by the intensity of the immunostaining of GnRH neurons and fibers in the brain and the amount of cfGAP in the pituitary. Treatment with KT, the main circulating androgen in adult male catfish, neither changed the number of cfGnRH neurons, nor elevated the cfGnRH content in the pituitary. In previous experiments with younger, prepubertal fish (2-6 weeks ph), T caused an elevation of the number of cfGnRH neurons to the same level as present in pubertal fish of 12-14 weeks. We conclude that the onset of puberty in the male African catfish coincides with the completion of the steroid-dependent structural maturation of the cfGnRH system in the brain. T and/or E(2), however, are still able to exert a positive influence on the amounts of cfGnRH during the later stages of pubertal development, thus still playing a role in the control of the cfGnRH system.     

Gonadotropin-releasing hormone II: a multi-purpose neuropeptide

Close to 30 forms of gonadotropin releasing hormone (GnRH) and at least five GnRH receptors have been identified in a wide variety of vertebrates and some invertebrates. One form, now called GnRH II, has the broadest distribution and the most ancient and conserved phylogeny. The distribution of the neurons that produce this peptide are completely nonoverlapping with any other GnRH forms. Fibers that project from these neurons overlap with GnRH I cells and/or fibers in a few regions, but are primarily divergent. The musk shrew (Suncus murinus) continues to be the most tractable mammalian species to use for studies of the function of GnRH II. The brain of the musk shrew has two GnRH genes (I and II), two GnRH receptors (types-1 and -2), and two different behaviors can be influenced by central infusion of GnRH II, but not by GnRH I; receptivity and feeding. Here, we summarize research on the musk shrew relative to the behavioral functions of GnRH II. First, female musk shrews are continually sexually receptive by virtue of their lack of an ovarian and/or behavioral estrus cycle. This feature of their reproductive ecology may be related to their semi-tropical distribution and their breeding season is highly dependent on changes in the availability of food. When food is not abundant, females stop mating, but brief bouts of feeding reinstate reproductive behavior. Likewise, intake of food is related to GnRH II mRNA and peptide content in the brain; after mild food restriction both decline. When GnRH II is infused centrally, at times when its content is low, it can both enhance receptivity and inhibit food intake. Simultaneous administration of a type-1 antagonist does not change the effect of GnRH II and use of an analog (135-18) that is a specific GnRH II agonist as well as a type-1 antagonist has the same effect as the endogenous GnRH II peptide. We propose that GnRH II plays a critical role by orchestrating the coordination of reproduction with the availability of nutritional support for these activities. Humans are bombarded with copious nutritional opportunities and at present obesity is a larger threat to health in many parts of the world than is under nutrition. It is our hope that understanding neuropeptides such as GnRH II that regulate food intake can ultimately lead to products that may curb appetite and thus decrease obesity and related risks to health.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.     

多马胺能药物对鲇鱼促性腺激素（GtH）分泌活动的影响

以珠江流域鲇鱼（silurus asotus)为实验材料,研究了多巴胺（DA）能药物（DA及其D－2型受体拮抗物 ,DOM）对鲇鱼促性腺激素（GtH)释放的影响,结果表明,在性腺发育的各个时期,单独注射DOM（5ug/g)均不能显著提高鲇鱼血液基础GtH水平,当DOM与LHRH－A联合注射时能显著增强LHRH－A刺激GtH释放的作用;DA只能抑制GnRH诱导的GtH释放,对基础GtH释放无抑制作用,这种生殖内分泌调节方式与鲇形目的革胡子鲇（Clarias gariepinus)和大鳍Hu（Mystus macropterus)相似,而与鲤形目的鲁科（Cyrpindiae)鱼类不同。     

Three forms of gonadotropin-releasing hormone,including a novel form,in a basal salmonid,Coregonus clupeaformis

Multiple forms of GnRH within individual brains may have different functions. However, some vertebrates such as salmonids continue to reproduce even though they have lost or do not express 1 of the 3 forms of GnRH found in most other teleosts. We examined a basal salmonid, lake whitefish, to determine the mechanism by which a reduction in the number of GnRH forms occurs. We identified for the first time 3 distinct GnRHs in a salmonid. One form is novel and is designated whitefish GnRH. The primary structure is pGlu-His-Trp-Ser-Tyr-Gly-Met-Asn-Pro-Gly-NH(2). HPLC and RIA were used for purification followed by Edman degradation for sequence determination. Mass spectroscopy was used to confirm the sequence and amidation of the peptide. The other 2 forms, salmon GnRH and chicken GnRH-II, are identical to the 2 forms found in salmon, which evolved later than whitefish. Synthetic whitefish GnRH is biologically active, as it increased mRNA expression of growth hormone and the alpha-subunit for LH and thyroid-stimulating hormone in dispersed fish pituitary cells. Our data support the hypothesis that the ancestral salmonid had a third GnRH form when the genome doubled (tetraploidization), but the third form was lost later in some salmonids due to chromosomal rearrangements. We suggest that the salmon GnRH form compensated for the loss of the third form.     

Neurosecretory neurons of the nucleus preopticus (NPO) express salmon GnRH mRNA and show reproduction phase-related variation in the female Indian major carp, Cirrhinus cirrhosus

We studied the expression of sGnRH mRNA in the neurons of the nucleus preopticus (NPO) of the Indian major carp, Cirrhinus cirrhosus, and their correlation with the reproductive status of the fish. Non-radioisotopic in situ hybridization histochemistry protocol employing biotinylated-oligonucleotide probes complementary to salmon GnRH, cichlid GnRH I, catfish GnRH, chicken GnRH II (from cichlid and catfish), and mammalian GnRH, were applied to the sections through the POA of the female Indian major carp Cirrhinus cirrhosus. Incubation with the probe complimentary to salmon GnRH (sGnRH) mRNA from salmon, produced distinct hybridization signal in the cytosol of several neurosecretory neurons of the magnocellular and parvocellular subdivisions of the NPO of the fish collected during February-April (preparatory phase) and May-June (prespawning phase). However, no signal was detected in the NPO of fish collected during July-August (spawning phase). Application of other antisense probes, or sense probe for salmon GnRH mRNA, produced no signal. We suggest that NPO neurons in C. cirrhosus may express sGnRH mRNA, produce GnRH peptide, and play a role in regulation of pituitary-ovary axis.     

Purification and characterization of iron superoxide dismutase and copper-zinc superoxide dismutase from Acanthamoeba castellanii

Two superoxide dismutases (SOD I and SOD II) were purified from Acanthamoeba castellanii and characterized for several biochemical properties. Analysis of the primary structure and inhibition studies revealed that SOD I is iron SOD (Fe-SOD), with a molecular mass of 50 kDa, and SOD II is copper-zinc SOD (Cu,Zn-SOD), with a molecular mass of 38 kDa. Both enzymes have a homodimeric structure consisting of 2 identical subunits, each with a molecular mass of 26 and 19 kDa for SOD I and SOD II, respectively. The isoelectric points of SOD I and SOD II were 6.4 and 3.5, respectively, and there were no isoenzyme forms detected. Both enzymes show a broad optimal pH of 7.0-11.0. Because no differences were observed in the apparent molecular weight of SOD I after addition of the reducing agent 2-mercaptoethanol, the subunits do not appear to be linked covalently by disulfide bonds. However, the subunits of SOD II were covalently linked by intra- and interdisulfide bonds. Western blot analyses showed that the 2 enzymes have different antigenicity. Both enzymes occur as cytoplasmic and detergent-extractable fractions. These enzymes may be potential virulence factors of A. castellanii by acting both as antioxidants and antiinflammatory agents. These enzymes may be attractive targets for chemotherapy and immunodiagnosis of acanthamoebiasis.     

GnRHs and GnRH receptors

GnRH is the pivotal hypothalamic hormone regulating reproduction. Over 20 forms of the decapeptide have been identified in which the NH2- and COOH-terminal sequences, which are essential for receptor binding and activation, are conserved. In mammals, there are two forms, GnRH I which regulates gonadotropin and GnRH II which appears to be a neuromodulator and stimulates sexual behaviour. GnRHs also occur in reproductive tissues and tumours in which a paracrine/autocrine role is postulated. GnRH agonists and antagonists are now extensively used to treat hormone-dependent diseases, in assisted conception and have promise as novel contraceptives. Non-peptide orally-active GnRH antagonists have been recently developed and may increase the flexibility and range of utility. As with GnRH, GnRH receptors have undergone co-ordinated gene duplications such that cognate receptor subtypes for respective ligands exist in most vertebrates. Interestingly, in man and some other mammals (e.g. chimp, sheep and bovine) the Type II GnRH receptor has been silenced. However, GnRH I and GnRH II still appear to have distinct roles in signalling differentially through the Type I receptor (ligand-selective-signalling) to have different downstream effects. The ligand-receptor interactions and receptor conformational changes involved in receptor activation have been partly delineated. Together, these findings are setting the scene for generating novel selective GnRH analogues with potential for wider and more specific application.     

Differential regional distribution of gonadotropin-releasing hormones in amphibian (clawed toad, Xenopus laevis) brain

In most vertebrate species two forms of gonadotropin-releasing hormone (GnRH) are present in the brain, and their differential distribution suggests they have different functional roles. The regional distribution and relative concentrations of GnRH molecular forms in the brain of adult clawed toad (Xenopus laevis) were determined using high performance liquid chromatography and radioimmunoassay with a library of region-specific GnRH antisera. Four immunoreactive forms of GnRH were detected: mammalian, hydroxyproline mammalian, chicken II, and an unidetified form of GnRH. Mammalian GnRH was distributed throughout the brain, and hydroxyproline mammalian was present in the forebrain, midbrain (excluding hypothalamus), and hypothalamus. Chicken GnRH II also occurred throughout the brain, but was present in greater amounts in the hindbrain and midbrain (excluding hypothalamus). An unidentified form of GnRH with properties of salmon GnRH was detected in the forebrain. Considering the relative proportions of mammalian GnRH and chicken GnRH II in the major brain areas, the concentration of mammalian GnRH was high in the forebrain, midbrain (excluding hypothalamus), and in particular in the hypothalamus, and very little chicken GnRH II was present in these areas. In the hindbrain, chicken GnRH II predominated and the concentration of chicken GnRH II was highest in the medulla. These findings suggest: (1) mammalian GnRH is the prime regulator of gonadotropin release from the pituitary, and (2) chicken GnRH II has an extrapituitary role.     

Catfish liver acid phosphatases: differently glycosylated enzyme molecules with altered kinetic properties

Two forms of catfish liver acid phosphatase (AcPase I and II) were separated and purified to homogeneity and their carbohydrate compositions and some biochemical properties were studied. Evidence is given that AcPase I and II are differently glycosylated forms of the same enzyme. The enzyme forms differ significantly in the size and the composition of their carbohydrate components, sensitivity towards sulfhydryl-blocking and protecting reagents, sensitivity to ferric and ferrous ions, thermostability and ability to hydrolyze some nucleotides. The more highly glycosylated form is more sensitive to thermal denaturation. AcPase I and II behave differently towards ascorbate and changes in its concentration and it is suggested that the concentration of reducing modifiers may regulate AcPase activity at the cellular level. It is hypothesized that the differing extents of glycosylation influence the structure of the enzyme forms. This is expressed in altered conformations of two enzyme forms and results in a different exposure of the essential cysteine residues.