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We investigated the mortality rates of male and female amphipods resulting from stickleback fish Culaea inconstans) and dragonfly nymphs (Basiaeschna janata) predation. Both predators consumed significantly more ovigerous female amphipods than non-ovigerous females or males in 24-h trials. Stickleback fish consumed more non-ovigerous females than males, whereas dragonflies consumed more males than non-ovigerous females. These results were consistent across trials conducted under ambient light-dark regimes and those conducted in the absence of light, although overall mortality rates were highest in light trials. There were no significant differences in the recorded behaviors of the two predators. However, the behavior of ovigerous females differed significantly from that of males and non-ovigerous females, which may help to explain the overall differences in mortality. These results suggest that female amphipods incur a significant cost of increased predation risk associated with maternal care.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor–induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as glut2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development. Copyright © 1997 Elsevier Science, Inc.

Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states. In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells. This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression. However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC). After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures. These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

We have investigated the role of hepatocyte growth factor/scatter factor (HGF/SF) in the growth and/or differentiation of pancreatic islet beta-cells. We found that in the human fetal pancreas immunoreactive HGF/SF receptor (c-met proto-oncogene product) is preferentially associated with the developing beta-cells. In the adult pancreas, c-met messenger RNA is highly enriched in the islets and the immunoreactive protein is also restricted to the islet beta-cells. HGF/SF messenger RNA content of fetal pancreas-derived fibroblasts is more than 10-fold higher than that of adult fibroblasts. Culture of human fetal pancreatic epithelial cells in conditioned medium from the fetal pancreatic fibroblasts caused a 2.4-fold stimulation of the formation of islet-like cell clusters that was due to both mitogenic and morphogenic effects. Beta-cell proliferation in the cell clusters was stimulated 3.5-fold by the conditioned medium, and this was associated with a marked decrease in insulin content. All of the effects of the conditioned medium were blocked by anti-HGF/SF antibody. Specificity was confirmed by overriding the blocking effect of the antibody with excess recombinant HGF/SF. Conditioned medium from adult pancreatic fibroblasts stimulated islet-like cell cluster formation only slightly, and did not affect beta-cell replication. These results suggest that HGF/SF secreted by fetal fibroblasts is mitogenic to beta-cells. Taken together, our findings indicate an important role for HGF/SF in fetal mesenchyme-induced pancreatic beta-cell growth.

Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.

One of the major β-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with α- and β-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.

The effects of nicotinamide (NIC) on human fetal and adult endocrine pancreatic cells were studied in tissue culture. Treatment of the fetal cells with 10 mM NIC resulted in a twofold increase in DNA content and a threefold increase in insulin content. This was associated with the development of beta cell outgrowths from undifferentiated epithelial cell clusters and an increase in the expression of the insulin, glucagon, and somatostatin genes. DNA synthesis was stimulated only in the undifferentiated cells. Half-maximal doses for the insulinotropic and mitogenic effects of NIC were 5-10 and 1-2 mM, respectively. Islet-like cell clusters cultured with NIC responded to glucose stimulation with a biphasic increase in insulin release (fourfold peak), whereas control cells were unresponsive to glucose. Both control and NIC-treated cells developed into functional islet tissue after transplantation into athymic nude mice. As compared with adult islets, the insulinotropic action of NIC could only be demonstrated in the fetal cells. Our results indicate that NIC induces differentiation and maturation of human fetal pancreatic islet cells. This model should be useful for the study of molecular mechanisms involved in beta cell development.

Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.

S: Differential developmental regulation of pancreas-specific genes has not been reported for the human fetal pancreas. We have therefore undertaken a systematic, quantitative analysis of the transcriptional levels of various genes in the human pancreas at different stages of fetal and postnatal development. Using sensitive ribonuclease protection assays, in situ hybridization, and the polymerase chain reaction, our results indicate the following: 7) Transcriptional levels of insulin and amylin remain lower in the fetal than in the adult pancreas, whereas glucagon and somatostatin mRNA levels are consistently greater after 14 wk gestation than postnatally. These results are in agreement with previous immunohistochemical studies of these gene products. 2) The reg gene exhibits a 20-fold increase in mRNA levels after 16 wk gestation. The gene is expressed exclusively in the acinar cells and does not colo-calize with insulin. This restricted exocrine expression does not indicate a direct role for the reg gene in islet development. 3) Glucose transporter 2 and glucokinase mRNA are detectable as early as 13 wk gestation and remain low throughout development. Glucose transporter 1 reaches adult transcriptional levels by 18 wk gestation. The early detection of glucose transporter 2 and glucokinase implies that lack of expression of these “glucose sensor” genes does not account for the known insensitivity of the fetal β-cells to glucose.

Reg is a gene associated with regeneration of pancreatic islets. We have previously shown that nicotinamide induces differentiation of human fetal β-cells in tissue culture and that hepatocyte growth factor/scatter factor (HGF/SF) is mitogenic for the fetal β-cells. We now tested whether these conditions, supporting either differentiation or growth, are associated with changes in reg gene expression in human fetal pancreatic cells. Culture for 7 days with 10 mM nicotinamide led to a fourfold decrease in reg mRNA levels (23 ± 12% of control, n = 5, P < 0.001). In contrast, HGF/SF increased reg expression threefold (302 ± 68% of control, n = 4, P < 0.05). Nicotinamide, which does not alter the differentiation level of adult β-cells, did not significantly affect reg expression in adult human islets (84 ± 4% of control, n = 2, NS). Thus, a higher level of endocrine differentiation is associated with a lower level of reg expression, and a higher rate of β-cell replication results in increased reg transcription. These results provide the first evidence of a molecular marker, the reg gene, to distinguish between proliferation and differentiation of human β-cells.

The production of human monoclonal antibodies is limited by a lack of good human fusion partners, the short-term stability of most human hybridomas, low fusion frequencies, and low secretion levels. An alternative method of immortalizing human immunoglobulin-secreting lymphocytes is presented here, which avoids many of the drawbacks associated with human hybridoma technology. Brief exposure of cells to a high intensity electric field results in the reversible permeabilization of cell membranes (electroporation). This technique allows the introduction of genes into virtually any cell type in a highly efficient manner. Specific genes or DNA sequences can thus be transferred into cells via the transient pores created in the membrane and, if the sequences are expressed, can yield cells with altered phenotypic properties (i.e., drug resistance). Electroporation of activated human lymphocytes in the presence of DNA sequences coding for immortalization functions and specifically targeted to B cells, would yield, via the stable integration of exogenous DNA into the cellular genome and the subsequent expression of the genes, immortalized, immunoglobulin-secreting lymphocytes, without the need for a fusion partner. Details of this technique, in addition to its advantages over the commonly used methods for generating hybridomas, are discussed.

Fibrpblast growth factors (FGFs) play an important role in the embryogenesis of many organs. In order to elucidate the regulation of pancreatic islet development, we have used tissue culture of human fetal pancreas to study the effects of FGFs. Our method is based on the formation of islet-like cell clusters (ICCs), consisting of 87% undifferentiated cells and 13% islet hormone-positive cells. Supplementation of the culture medium with FGF-2 (basic FGF) more than doubled the formation of ICCs, stimulated their DNA synthesis, inhibited the insulin release (ED50-1 ng/ml), but did not affect the insulin content. This activity was shared by FGF-1 (acidic FGF) but not by FGF-6 or FGF-7 (KGF). When ICCs were treated for 72h with bFGF-Saporin (F-SAP), a mitotoxin targeted against FGF-receptor expressing cells, a distinct population (45%) of cells within the ICCs were killed, whereas some strongly hormone-positive (mature) islet cells were left intact. F-SAP treatment also blocked the endocrine differentiation induced by nicotinamide. Similar experiments with isolated adult human islets showed no activity of FGF-2 or F-SAP, supporting a role for FGF in the early development of human islet cells.

Isolation of the endocrine cell precursors from the human fetal pancreas is important for the understanding of islet cytodifierentiation. The progenitor cells, from which all four islet endocrine cell types arise, are present within the epithelium ol the fetal pancreatic duct. Following enzymatic digestion and culture of the fetal pancreas WG obtained cell clusters resembling islets but with a high content of undifferentiated cells. Histochemical staining revealed very high acid β-galaclosidase activity in most cells within the clusters. After transplantation into athymic nude mice, the clusters gave rise to tissue rich in differentiated endocrine cells. The histochemical finding of high acid β-galactosidase activity was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid β-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic β-galactosidase substrate, we were able to enrich for a subpopulation of cells high in acid β-galactosidase activity with flow cytometry. Evidence identifying these cells as potential islet cell precursors included, besides the transplantation experiments, the colocalization in vitro of lyrosine hydroxylase, a known marker of embryonic islet cells. Thus, our results indicate that high acid β-galactosidase activity serves as a marker to enrich populations of endocrine cell precursors.

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)

The differential expression of a series of proto-oncogenes has been examined in a group of cultured murine T-cell lymphomas that were induced following virus inoculation into, or X irradiation of, C57BL/6 mice. Two classes of T lymphoma cell lines were studied: growth factor-dependent (autocrine) cells, and growth factor-independent T lymphoma cells. Cell lines that were established from X-irradiation-induced T lymphomas were growth factor dependent, whereas T lymphoma lines grown from virus-induced tumors were generally growth factor independent. Of 18 cellular proto-oncogenes studied, five (c-myc, c-myb, c-abl, c-rasHa, c-rasKi) were consistently expressed in all cell lines tested. Thirteen other proto-oncogenes (c-mos, c-sis, c-rel, c-yes, c-fes3, c-fes4, c-fos, c-fms, c-src, c-erbA, c-erbB, int-1, Pim-1) were not expressed in any of the T lymphoma cells tested. Concanavalin A-stimulated spleen cells, representative of replicating T cells, expressed c-myc, c-abl, and Pim-1, suggesting that the products of these proto-oncogenes are involved in T-cell proliferation. The results indicate no qualitative differences (albeit some quantitative differences) in proto-oncogene expression between the growth factor-dependent and growth factor-independent cells. This suggests that expression of the five proto-oncogenes is in itself not sufficient to induce the progression of the growth factor-dependent cells to their fully growth factor-independent counterparts. Changes in the regulation of one or more of the five active proto-oncogenes, i.e., from an inducible to a constitutive state, and/or additional changes in the expression of other cellular genes may be required to induce the transformation of neoplastic T cells from growth factor dependence to growth factor independence.

The effects of cis-[Pt(NH3)2Cl2] on the spontaneous cell-mediated cytotoxicity of human lymphoblast cell lines mediated by normal human peripheral blood lymphocytes was investigated. Direct addition of cis-[Pt(NH3)2Cl2], but not its tumor inactive trans isomer, to the in vitro spontaneous cytotoxicity assay resulted in a small but significant enhancement of target cell lysis. Pretreatment of target cell lines with 5 micro M cis-[Pt(NH3)2Cl2] also resulted in a similar increase in target cell susceptibility to lysis.

Human lymphoblast cell lines (LCL) were treated with the antitumor complex Cis-[pt(NH3)2Cl2] (CPDC). Direct effects of CPDC treatment on LCL growth, as well as on LCL recognition by human lymphocytes in vitro are reported. Treatment of LCL with 10 micro m CPDC strongly inhibits DNA and protein synthesis, but has little effect on LCL viability. At this concentration, CPDC also inhibits the proliferation of human peripheral blood lymphocytes (PBL) in response to mitogens or allogeneic cells. Therefore, studies of LCL recognition by human PBL were conducted using CPDC pretreated and extensively washed LCL. Recognition of CPDC pretreated LCL in the in vitro mixed lymphocyte tumor (MLT) cell assay for the activation phase of cellular immunity is reduced. The decrease in MLT response is not due to direct effects of the complex on the responding T lymphocytes, but may be due to lowered antigen expression of treated LCL or other mechanisms. If CPDC does enhance tumor rejection in vivo, it does not appear to be due to enhanced recognition by T cells.

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)