The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAA-CAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherchia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 12'I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepi-

Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.Pathogenic strains of E. coli and other bacteria produce a family of heat-stable enterotoxins (STs) that activate intestinal guanylate cyclase. STs are acidic peptides that contain 18 or 19 amino acids with six cysteines and three disulfide bridges that are required for full expression of bioactivity (6). The increase of intestinal epithelial cyclic GMP elicited by STs is thought to cause a decrease in water and sodium absorption and an increase in chloride secretion (7,8). These changes in intestinal fluid and electrolyte transport then act to cause secretory diarrhea. In developing countries, the diarrhea resulting from STs causes many deaths, particularly in the infant population (9). STs are also considered a major cause of traveler's diarrhea in developed countries (10). They have also been reported to be a leading cause of morbidity and death in domestic animals (11).In the present study, we designed a bioassay to search for a potential endogenous ligand that activates the intestinal guanylate cyclase. This bioassay is based on the demonstration that T84 cells in culture respond to ST in a selective and sensitive manner with graded increases of intracellular cyclic GMP. This bioassay revealed that the intestine as well as the kidney possessed an active material. Purification of this material from the rat intestine was accomplished and the structure was determined to be a 15-amino acid peptide with 4 cysteines t...

The human malaria parasite, Plasmodium falciparum, de-grades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy. (J. Clin. Invest.

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins (ST), is an endogenous activator of guanylate cyclase C (GC-C). We have initiated a search for other members of the guanylin peptide family and in the current study describe a "guanylin-like peptide" from human urine. Bioactivity was monitored by determining the effect of urine extracts on T84 cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. Purification yielded two bioactive peaks of peptides that, when sequenced by NH2-terminal analysis, possessed 15 and 16 amino acids. The sequence of the smaller peptide represented an NH2-terminal truncation of the larger peptide. We have termed the larger peptide human uroguanylin; it has the following amino acid sequence: NDDCELCVNVACTGCL. Human uroguanylin shares amino acid sequence homology with guanylin and ST. Synthetic uroguanylin increased cGMP levels in T84 cells, competed with 125I-labeled ST for receptors, and stimulated Cl- secretion as reflected by an increased short-circuit current. Thus we report the isolation from human urine of a unique peptide, uroguanylin, that behaves in a manner similar to guanylin and appears to be a new member of this peptide family.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin of the cellular proteins are Na-acetylated (7). As for the remaining free-N-termini proteins, the rules that govern removal of the initiator methionine residue by methionine aminopeptidase suggest that in most cases this residue is cleaved only when the penultimate residue is a "stabilizing" amino acid (8). Thus, proteins with exposed destabilizing N-termini appear to be sparse. (ii) The ubiquitin system degrades Na-acetylated proteins in a process that does not require removal ofthe modifying group and exposure ofa free N-terminal residue (9). We have previously shown that the degradation of certain Na-acetylated proteins requires a specific factor that is not required for the breakdown of free N-termini proteins. The factor, designated factor Hedva (FH), is required for the proteolysis of the core nucleosomal histone H2A, the cytoskeletal protein actin, and the lens protein a-crystallin (11). FH is a homodimer with a subunit molecular mass of46 kDa. Initial analysis of the mechanism of action of FH revealed that it is not involved in the conjugation process. Rather, it acts along with the 26S protease complex and stimulates degradation of conjugated H2A. The effect appears to be specific to this group of proteins, as the factor is not required for the degradation ofconjugates of several proteins with free N-termini, such as oxidized RNase A and lysozyme (11). Further analysis demonstrated that FH probably interacts with the conjugates prior to their degradation: incubation of conjugates in the presence of purified FH and the protease revealed a short, but significant, time lag that preceded initiation of degradation. The lag was completely abolished when FH was preincubated with the conjugates prior to the addition of the protease. These findings demonstrate that recognition of certain proteins and their targeting for degradation involves both conjugation ofubiquitin and degradation of the adducts by the 26S protease complex. MATERIALS AND METHODSPreparation of Ubiquitin-Conjugated Histone H2A. Multiply ubiquitinated histone H2A was prepared using 125I-H2A, ubiquitin, ATP, and purified El (ubiquitin-activating enzyme), E2 (14-kDa ubiquitin-carrier protein or ubiquitinconjugating enzyme), and E3a (ubiquitin-protein ligase) as described (11).