The metabolism of fatty acids in human Bietti crystalline dystrophy.

PURPOSE: To investigate the role of abnormal lipid metabolism in Bietti crystalline dystrophy. METHODS: Cultured human lymphocytes and fibroblasts from patients with Bietti crystalline dystrophy (BCD) were incubated in the presence of [(14)C]18:3n-3 or [(14)C]18:2n-6. Incorporation into the cellular lipid pools and further metabolism by desaturation or elongation were monitored by thin-layer chromatography and HPLC. Results were compared with those in normal control subjects and patients with Wolman disease (WD). RESULTS: Pulse-chase experiments with labeled fatty acids in all groups showed that, after 1 hour, radioactivity was largely confined to the triacylglyceride (TG) and choline phosphoglyceride (CPG) pools. However, after several hours, radioactivity was transferred from the TG and CPG pools, some going to the serine and ethanolamine phosphoglyceride (SPG and EPG) pools. Fibroblasts from all groups showed direct transfer of fatty acids (FAs) into CPG and EPG. Incorporation of labeled FAs into the EPG pool paralleled extensive desaturation and elongation of 18:2n-6 to 22:5n-6 and 18:3n-3 to 22:6n-3. Fibroblasts from patients with WD (a lysosomal acid lipase deficiency characterized by excessive lipid accumulation), showed higher incorporation of 18:2n-6 into TGs than did normal or BCD fibroblasts. Conversely, fibroblasts from patients with BCD showed lower conversion of 18:3n-3, but not of 18:2n-6, into polyunsaturated FAs (PUFAs) than those of normal subjects or patients with WD. This was true for total FAs, CPGs, and EPGs. Similar results were found in both fibroblasts and lymphocytes; however, unlike fibroblasts, lymphocytes from normal subjects showed similar levels of incorporation of FAs into EPGs and CPGs. In contrast, incorporation of 18:3n-3 into EPGs was decreased in lymphocytes from patients with BCD. CONCLUSIONS: BCD is characterized by a lower than normal conversion of FA precursors into n-3 PUFA, whereas there is a higher than normal level of n-6 and n-3 FAs incorporation into TGs in cells from patients with WD. These findings raise the possibility that abnormal lipid metabolism associated with BCD is the result of deficient lipid binding, elongation, or desaturation in contrast to the lysosomal acid lipase deficiency found in Wolman disease.

Oxidative stress induces heme oxygenase-1 immunoreactivity in Müller cells of mouse retina in organ culture.

PURPOSE: Heme oxygenase (HO)-1 immunoreactivity (IR) was examined in normal untreated retina and in retinal explants after in vitro treatment with stress agents. METHODS: Enucleated eyes from young adult C3H mice were immediately fixed and cryosectioned and the retina sections processed for immunocytochemistry with antibodies against HO-1 and glial fibrillary acidic protein (GFAP). From other eyes retinas were isolated and maintained in organ culture, either untreated for 4 days maximum or for 21 hours during which the explants were treated the first 3 hours with selected doses of sodium arsenate or hydrogen peroxide. Thereafter, the explants were processed identically with the normal tissue. RESULTS: In the normal retina, HO-1 and GFAP IR was very low. The culturing itself resulted in an increase in both HO-1 and GFAP immunolabeling in Müller cells of explanted retinas. Both sodium arsenate and hydrogen peroxide further induced strong HO-1 IR in Müller cells but not in other retinal cells. In contrast to HO-1, GFAP staining in Müller cells was not altered as a result of treatment, either by sodium arsenate or hydrogen peroxide at any concentration used. CONCLUSIONS: The results show for the first time that HO-1 can be induced in the retina in vitro by conditions of oxidative stress and that enzyme expression is confined exclusively to Müller cells.

Clusterin protein diversity in the primate eye.

PURPOSE: The clusterin gene encodes a multi-functional protein that has been identified in different tissues, including a number of different eye tissues, primarily in the mouse and to a much lesser extent in humans. Clusterin has been implicated in a number of cellular processes such as lipid transport, membrane integrity, apoptosis, and neurodegeneration, all of which could be important to the biology of the eye. In the current communication, we provide data that confirms the expression of clusterin in a number of different human eye tissues and establishes the expression profile of this gene in monkey derived eye tissues. The issue that we sought to examine is whether a broad profile of clusterin expression in the eye is consistent in primates (monkey and human). METHODS: The majority of our study was done using monkey eye tissues. Where possible, we have used human tissues in order to confirm published findings. Northern and western analysis was performed using tissues derived from monkey eyes. In situ hybridization and immunochemistry were carried out on human eye sections. RESULTS: Clusterin mRNA is expressed in primate lens, cornea, limbus, sclera, orbital muscle, ciliary body, retina, RPE/choroid, and RPE cells in culture. Western analysis revealed that two major groups of clusterin exist in the eye, a high molecular weight group (>100 kDa) and a second group consisting of at least five clusterin species that are all approximately 80 kDa. Analysis of conditioned media from RPE cells cultured on permeable supports suggests that different forms of clusterin display alternative patterns of secretion. CONCLUSIONS: Clusterin is expressed in a broad range of eye tissues in both human and monkey, suggesting that this is a characteristic feature in primates. We demonstrate for the first time that a diverse number of clusterin isoforms were observed in monkey eye tissues by western analysis. Meanwhile, the molecular size of clusterin mRNA detected in the array of tissues are identical in size, suggesting that the nature of the diversity in clusterin forms is due to post-translational modifications. In addition, new insights were made in defining clusterin expression in ciliary body, cornea, and the retinal pigment epithelium.

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor activate cellular metabolism and inhibit proliferation of the RAW macrophage cell line.

Recombinant forms of the neurotrophic factor pigment epithelium-derived factor (PEDF) activate metabolism of RAW macrophage cells while simultaneously inhibiting their proliferation. The recombinant forms (rPEDF) acted with EC(50)s of 0.1-1 nM while full-length native bovine PEDF was inactive. Urea, which is the buffer used to extract recombinant PEDF, stimulated RAW cell proliferation, the first report of an effect of urea on non-kidney cells. PEDF acted within 12 h and its effects persisted up to 72 h with continuous exposure. Although rPEDF had no direct action on glioma cell lines, it increased the amount of a soluble factor released by RAW cells which was capable of blocking glioma cell division. Thus PEDF may function as a neuroimmune modulator, affecting both neural and immune system cells.

Identification, isolation, and characterization of a 32-kDa fatty acid-binding protein missing from lymphocytes in humans with Bietti crystalline dystrophy (BCD).

Bietti crystalline dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by intraretinal lipid inclusions with degeneration of the retina and sclerosis of the choroidal vessels, resulting clinically in progressive night blindness and constriction of the visual fields. Characterization of fatty acid metabolism in Bietti crystalline dystrophy suggested that BCD might result from abnormalities in lipid-binding proteins or one or more enzymes active in fatty acid elongation and desaturation. To further investigate the first possibility, the docosahexaenoic acid-binding proteins (DHABPs) of human lymphocytes from patients with Bietti crystalline dystrophy were studied and compared with those of normal controls. For fatty acid-binding protein (FABP) identification, lymphocyte cytosol was first subjected to Lipidex 1000 chromatography. FABPs were then cross-linked with [14C]22:6n-3 and identified by HPLC and SDS-PAGE. Ten major peaks corresponding to calculated molecular weights of 13, 14, 32, 43, 45, 50, 64, 96, 105, and 186 kDa exhibit high-affinity binding of fatty acids. Significantly, peaks corresponding to two fatty acid-binding proteins of 32 and 45 kDa present in age-matched controls are absent from lymphocytes of patients with BCD. The 32-kDa fatty acid-binding protein present in normal individuals but absent from patients with BCD was isolated from cultured control human lymphocytes, its fatty acid-binding properties were characterized, and its amino acid composition was analyzed. It shows specific binding of 3n-3 fatty acids, consistent with the pattern of abnormalities of lipid metabolism demonstrated in patients with BCD. These results suggest that the 32- and 43-kDa FABPs are reasonable candidates for causing BCD.

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.

We have shown previously that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed "natural" apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum- and 25 mM KCl-containing medium to serum-free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine-protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis.

Structural and comparative analysis of the mouse gene for pigment epithelium-derived factor (PEDF).

PURPOSE: Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mouse PEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mouse PEDF cDNA was cloned from a mouse liver cDNA library using human PEDF cDNA as a probe. The mouse PEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mouse PEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mouse PEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and human PEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.

Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes.

Pigment epithelium-derived factor (PEDF), a neurotrophic agent first identified in conditioned medium from cultured human retinal pigment epithelial cells, induces neuronal differentiation with neurite outgrowth in Y-79 retinoblastoma cells and has a neurotrophic survival effect on cerebellar granule cells in culture. In the present study, we investigated the effects of human recombinant PEDF (rPEDF) on proliferation and activation of microglia and astrocytes isolated from newborn rat brain. rPEDF treatment caused microglia to round up morphologically, increased their metabolic activity (measured by both MTS conversion and acid phosphatase activity), but blocked proliferation (mitosis). This blocking effect could be demonstrated in cultures stimulated to proliferate by addition of granulocyte-macrophage colony stimulating factor. The effect of rPEDF on microglial metabolic activity showed a dose-response relationship both in serum-containing medium and in chemically defined medium and was blocked with anti-PEDF antibody. rPEDF had no direct effect on the metabolic activity or proliferation of cultured astrocytes but blocked their proliferation in astrocyte-microglia co-cultures. Proliferation of isolated astrocytes was also blocked by conditioned medium from microglia treated with PEDF (PMCM). The effect of PMCM on astrocytes was not blocked by an antibody to transforming growth factor-beta. These results demonstrate that PEDF activates microglial metabolism while blocking proliferation and suggest that a soluble factor(s) released by rPEDF-stimulated microglia blocks the proliferation of astrocytes. Thus, PEDF could play an important role in regulation of glial function and proliferation in the central nervous system.

Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors.

Deletion of 17p is the most frequent abnormality observed in central nervous system (CNS) primitive neuroectodermal tumors (PNETs), implicating the presence of a tumor suppressor gene which maps to 17p. The gene for pigment epithelium-derived factor (PEDF) has been cloned and mapped to 17p13. PEDF belongs to the serine protease inhibitor (SERPIN) gene family. The PEDF protein has neurotrophic and neuronal-survival activities and is expressed in the CNS. Twenty tumor and matched normal DNA samples from patients with PNETs were screened by single-strand conformation polymorphism (SSCP) analysis to determine loss of heterozygosity (LOH) and to identify potential mutations within the 8 exons of the PEDF gene. Ten of the 20 tumors demonstrated LOH, consistent with the deletion status of 17p determined by cytogenetic or fluorescence in situ hybridization studies. SSCP analysis of the genomic DNA from the 10 cases with LOH demonstrated several polymorphisms in exons 4 and 7, but no mutations. Our results are consistent with a loss of alleles on 17p in 50% of CNS PNETs, but do not suggest that PEDF is a candidate for the PNET suppressor gene in 17p13.

Human gene for the RNA polymerase II seventh subunit (hsRPB7): structure, expression and chromosomal localization.

The human gene for the seventh largest subunit of RNA polymerase II complex, hsRPB7 was cloned, sequenced and mapped. This complex is an integral part of the transcription-coupled DNA repair mechanism and has been shown to be involved in several human genetic diseases and implicated in many others. The hsRPB7 gene consists of 8 exons and spans approximately 5.1 kb. Southern blots of genomic and cloned DNA suggest that hsRPB7 is coded for by a single gene. Using human radiation hybrids and YACs, the gene was localized to 11q13.1, within 70 kb of marker D11S1765. The sequence of the 5' flanking region does not contain a TATA element, but does contain several Sp1 binding sites, an AP-1 site and a novel inverted polymorphic GATA tandem repeat. This novel GATA repeat can be used for linkage analysis. The hsRPB7 gene seems to be highly conserved among eukaryotic species, showing general sequence conservation to yeast and Drosophila. Northern blot analysis reveals a high degree of tissue-specific expression. For example, adult retina, brain and kidney exhibit a relatively high level of expression. A moderate level of expression is observed in heart, lung, testis, cornea, retinal pigmented epithelium/choroid and placenta with a lower level of expression in the uterus, small intestine and skeletal muscle. A very low level of expression was observed in stomach and liver. Comparison between four fetal and adult tissues also demonstrate a surprising level of developmental specificity. Expression in fetal retina is considerably lower than fetal brain but similar to adult retina.

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (beta1-4, gamma1-2, and delta1-2), we have found PLCbeta4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the alpha-subunits of the G(q) family of G proteins, which are known to activate PLCbeta4, have also demonstrated G(alpha11)-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCbeta4 and anti-G(alpha11) antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G(alpha11) or a G(alpha11)-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G(alpha q) (another G(q) family member), are present elsewhere in the retina.

Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity.

Pigment epithelium-derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 microM glutamate, glutamate-induced neuronal death was significantly reduced. The protective effect of rPEDF was dose-dependent in the range from 0.023 to 7.0 nM (1-500 ng/ml), with a half-maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate-induced neurotoxicity, possibly via a calcium-related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.

Organization, evolutionary conservation, expression and unusual Alu density of the human gene for pigment epithelium-derived factor, a unique neurotrophic serpin.

PEDF is a neurotrophic serpin that promotes a neuronal phenotype and augments neuronal cell survival. The isolation, sequence and structural analysis of the human PEDF gene and its promoter along with its evolutionary conservation and expression in human tissues are now described. The gene spans approximately 16 kb and is divided among 8 exons and 7 introns, the junctions of which conform to the AG/GT consensus rule. PEDF appears to fall into the ovalbumin/PAI-2 subgrouping of serpins and is structurally far different from GDN/PN-1, the only other neurotrophic serpin reported to date. The immediate 5'-flanking region is dominated by a dense cluster of Alu repeats in which are embedded several promoter consensus sequences. A CAAT box is present at -43. The putative promoter region is also far different from that reported for GDN/PN-1. Comparable hybridization signals of 23 kb EcoRI fragments containing the PEDF gene are observed by Southern blot analysis in all primate, mammal and avian species examined; conservation is particularly evident among the primates. Northern blot analysis confirms the presence of the PEDF transcript in a broad range of human fetal and adult tissues including almost all brain areas examined, underscoring differences with GDN/PN-1 which, in the adult brain, is only expressed in glia and a subset of neurons.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation.

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (approximately 2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole-sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Structural organization and chromosomal localization of the human ribosomal protein L9 gene.

The intron-containing gene for the human ribosomal protein L9 has been cloned, sequenced and localized. The gene is approximately 5.5 kb in length and contains 8 exons. Splice sites follow the AG/GT consensus rule. The message for human rpL9 is 712 nt in length and is detected in all tissues examined. In the adult, expression is highest in retina and liver while brain shows highest expression among the fetal tissues tested. The transcription start site contains an oligopyrimidine tract, TTCTTTCTT, similar to those found in other ribosomal protein genes. As in other previously characterized ribosomal protein genes, a TATA box is absent from the 5' flanking region but a number of elements recognized by common transcription factors are present including Sp1 sites, CACCC boxes, inverted CCAAT boxes, and GATA elements. Another possible element of interest in the rpL9 5' flanking region is RFX1 also found in the well characterized rat rpL30 promoter. The gene was mapped by fluorescent in situ hybridization to band 13p of chromosome 4. At least 8 possible pseudogenes are present in the human genome, one of which is on Xp. As assessed by Southern 'Zoo-blot' analysis and direct cDNA sequence comparison, the human ribosomal protein L9 gene, like other ribosomal protein genes, is highly conserved among mammals.

Ultrastructural and biochemical analysis of sperm flagella from an infertile man with a rod-dominant retinal degeneration.

This study examined the ultrastructural morphology and posttranslationally modified alpha-tubulin isoforms in the sperm flagella of a patient presenting with infertility and retinal degeneration. Clinical evaluation showed impaired motility and gross morphological abnormalities of the sperm and a rod-dominant retinal degeneration with midperipheral pigment clumping and scattered bone spicules. Other neurological indications included delayed neuroelectric transmission in the auditory brainstem and a temporal lobe seizure disorder. Ultrastructural analysis showed that 46% of sperm axonemes had missing and/or misplaced doublets compared with 10% to 12% in control subjects. ELISA analysis showed hypoacetylation of alpha-tubulin (30% of control) but normal levels of alpha-tubulin tyrosination. Tubulin acetyl-transferase specific activity was also 30% of control activity. These characteristics may be indicative of microtubule instability leading to the pathological consequences described.

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Identification of arylamine N-acetyltransferase activity in the bovine lens.

Arylamine N-acetyltransferase (NAT) activity was identified and partially characterized in the bovine lens. According to size-exclusion HPLC, the molecular mass of the arylamine NAT is approximately 30-kDa. Based upon substrate specificity analysis, it is best described as an arylamine NAT which has some ability to N-acetylate arylalkylamines. This arylamine NAT acetylates para-aminobenzoic acid thereby demonstrating a monomorphic pattern of N-acetylation. It demonstrates low sensitivity to methotrexate inhibition as indicated by the relatively high IC50 value (470 microM). NAT could be involved in lenticular detoxification of both endogenous amines and exogenous drugs.