Pelvic organ prolapse is associated with alteration of sphingosine-1-phosphate/Rho-kinase signalling pathway in human vaginal wall.

Pelvic organ prolapse (POP) is a debilitating condition of unknown aetiology affecting > 50% of women over 40 years of age. In POP patients, the vaginal walls are weakened allowing descent of pelvic organs through the vagina. We sought to determine if sphingosine-1-phosphate (S1P) signalling, which regulates smooth muscle contractility and apoptosis via the RhoA/Rho-kinase (ROK) pathway, is altered in the vagina of women with POP. Utilising anterior vaginal wall specimens, we provide novel demonstration of the S1P pathway in this organ. Additionally, comparing specimens from women having pelvic reconstructive surgery for POP and control subjects, we reveal increases in mRNA expression of the three major mammalian S1P receptors (S1P1-S1P3), and RhoA and the ROK isoforms: ROKα and ROKß in POP patients, which correlates with a decrease in elastic fibre assembly pathway constituents. Taken together, our data suggest the S1P/ROK pathway as a novel area for future POP research and potential therapeutic development.

Expression of myosin isoforms in the smooth muscle of human corpus cavernosum.

The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.

Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase beta.

Corpus cavernosum smooth muscle (CCSM) from rabbits made diabetic for 6 months as a result of alloxan injection exhibited increased sensitivity (3vs 9 nM EC(50)) and generated 20-50% greater force to endothelin-1 (ET-1) compared to CCSM from normal rabbits. In contrast, the force produced by the CCSM in response to KCl and phenylephrine was not significantly altered in diabetic CCSM. The increased ET-1 sensitivity is associated with a two to three-fold upregulation of ET receptor A at both mRNA and protein levels in diabetic CCSM. ET-1-induced CCSM contraction is largely dependent upon Rho-kinase (ROK), since it is almost completely blocked by Y-27632 (a highly selective ROK inhibitor). Furthermore, expression of ROKbeta isoform is selectively upregulated in CCSM from diabetic rabbits. Thus, an increased CCSM tone, modulated by sensitization of the endothelin-mediated contractile pathway via ROK, may be a key component of the molecular mechanism of diabetes-induced erectile dysfunction.

Heterogeneity of bladder myocytes in vitro: modulation of myosin isoform expression.

We studied the expression of myosin heavy chain isoforms differing at the N-terminal (SM-A, SM-B) and the C-terminal (SM1, SM2) regions and non-muscle myosin heavy chain II-A and II-B (NMMHC II-A and B) in newborn and adult rabbit bladder smooth muscle cells (SMCs) and in cultures of enzymatically dissociated neonatal detrusor. RT-PCR analyses revealed that 94.5+/-3.27% of MHC transcripts of the adult bladder SMCs contained the 21-nucleotide insert (SM-B) compared with 83.8+/-3.2% in the newborn bladder, with the remainder of the mRNA being non-inserted (SM-A). In 3, 7, and 10 days of primary culture (proliferating, confluent, and post-confluent, respectively) and up to 4 subculture passages, bladder myocytes expressed predominantly SM-A. Immunofluorescence microscopy revealed heterogeneity in cultured myocytes, i.e. SM-B positive cells coexisting with negatively stained cells. In adult bladder, the C-terminal isoforms SM1 and SM2 represented, 43.1+/-4.3% and 56.89 + 4.3% of the mRNA, respectively, while newborn bladders expressed 72.5+/-7% SM1 and 27.5+/-7% SM2. Upon culturing, cells predominantly expressed SM1 at both the mRNA and protein levels. NMMHC II-A was expressed by both adult and newborn bladders and in culture, whereas NMMHC II-B was expressed at low levels only in newborn bladders, but upregulated in culture. These data indicate that bladder myocytes in vitro undergo modulation with relative overexpression of SM-A and SM1 and upregulation of NMMHC II-B. Information on the mechanisms responsible for this modulation in vitro might provide an understanding of the nature of altered myosin isoform expression associated with smooth muscle dysfunction in certain bladder diseases.

Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle.

Urinary bladder filling and emptying requires coordinated control of bladder body and urethral smooth muscles. Bladder dome, midbladder, base, and urethra showed significant differences in the percentage of 20-kDa myosin light chain (LC20) phosphorylation (35.45 +/- 4.6, 24.7 +/- 2.2, 13.6+/- 2.1, and 12.8 +/- 2.7%, respectively) in resting muscle. Agonist-mediated force was associated with a rise in LC20 phosphorylation, but the extent of phosphorylation at all levels of force was less for urethral than for bladder body smooth muscle. RT-PCR and quantitative competitive RT-PCR analyses of total RNA from bladder body and urethral smooth muscles revealed only a slight difference in myosin heavy chain mRNA copy number per total RNA, whereas mRNA copy numbers for NH2-terminal isoforms SM-B (inserted) and SM-A (noninserted) in these muscles showed a significant difference (2.28 x 10(8) vs. 1.68 x 10(8) for SM-B and 0.12 x 10(8) vs. 0.42 x 10(8) for SM-A, respectively), which was also evident at the protein level. The ratio of COOH-terminal isoforms SM2:SM1 in the urethra was moderately but significantly lower than that in other regions of the bladder body. A high degree of LC20 phosphorylation and SM-B in the bladder body may help to facilitate fast cross-bridge cycling and force generation required for rapid emptying, whereas a lower level of LC20 phosphorylation and the presence of a higher amount of SM-A in urethral smooth muscle may help to maintain the high basal tone of urethra, required for urinary continence.

Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle.

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.

Improved contractility of obstructed bladders after Tadenan treatment is associated with reversal of altered myosin isoform expression.

PURPOSE: Tadenan is a plant extract from Pygeum africanum used in the treatment of benign prostatic hyperplasia, to protect the bladder from contractile dysfunction induced by partial bladder outlet obstruction (BOO). The aim of the present study was to determine whether the Tadenan-induced return of detrusor contractility affects the expression of myosin isoforms, which differ at the C-terminal (SM1 and SM2) and the N-terminal regions (SM-A and SM-B). MATERIALS AND METHODS: Four groups of New Zealand White rabbits (3 to 5 kg., 4 to 6 rabbits per group) were either partially obstructed by ligation of the urethra (groups 1 and 2) or not obstructed (groups 3 and 4). After 2 weeks, rabbits from groups 2 and 4 received Tadenan in peanut oil (vehicle) orally at 100 mg. /kg./day for 3 weeks and rabbits in groups 1 and 3 received vehicle only. Rabbits were sacrificed and bladders were removed and weighed. Contractility studies were performed on isolated strips of detrusor and the remaining muscular layer from the bladder body was used to study the expression of myosin heavy chain (MHC) isoforms at mRNA (SM1, SM2, SM-A, and SM-B) and the protein (SM1 and SM2) levels by RT-PCR and SDS-PAGE analyses, respectively. RESULTS: Tadenan significantly reduced the effect of BOO on bladder mass. The diminished contractile response to field stimulation and carbachol secondary to urethral obstruction was significantly reversed by Tadenan treatment. The relative ratios for MHC isoforms were altered at the mRNA (SM2:SM1 and SM-A:SM-B) and protein (SM2:SM1) levels in obstruction. Upon treatment with Tadenan, the ratio of these isoforms returned to normal, as shown at the mRNA levels. In addition, the altered relative ratio of SM2:SM1 at the protein level also returned to nearly normal values after treatment. CONCLUSIONS: Improvement of obstruction-induced contractile dysfunction of the detrusor following treatment with Tadenan is associated with changes in the expression of myosin isoforms. The alteration in the expression of myosin isoforms associated with obstruction-induced hypertrophy is reversed close to normal in the detrusor smooth muscle from Tadenan-treated obstructed rabbits.

Lower urinary tract physiology and pharmacology.

This review focuses on what we consider to be the most important findings of the last year relating to the smooth muscle of the lower urogenital system and the different levels of regulation that control its contraction and relaxation. One level is through modulation of the smooth muscle itself or its environment. Recent findings examining myosin isoform composition and collagen content as well as mechanisms that appear to be involved in inducing hyperplasia/hypertrophy of smooth muscle are described. Another method of regulation is via calcium-dependent phosphorylation of the regulatory light chain of myosin, which increases its activity. Interesting results indicating an uncoupling of force from calcium in the bladder are discussed. A third level of regulation is pharmacologic. Thus, the most recent findings related to receptor subtypes, including muscarinic, endothelin, alpha-adrenergic and nicotinic receptors, are presented. In addition, the effects of diabetes, incontinence, and partial bladder outlet obstruction on these modes of contractile regulation are also discussed.

Myosin light chain phosphorylation at resting level and the composition of myosin isoforms in the bladder body and urethra.

Bladder filling depends upon the coordinated control of a storage chamber, the bladder body, and its outlet, the bladder base and urethra. Bladder emptying results from development of force in the bladder body and relaxation of the outlet. Muscle strips from bladder body reveal phasic characteristics, whereas the strips from urethral wall are tonic. To determine whether the compositions of myosin heavy chain (MHC) isoforms and the level of myosin light chain (MLC) phosphorylation contribute to the regional variation in the contractile states of the bladder smooth muscle, we analyzed the levels of MLC phosphorylation and the expression of myosin isoforms in smooth muscle tissues from different regions of the urinary bladder. Strips of bladder from the dome, mid body, base of the bladder and urethra were removed and analyzed for the levels of MLC phosphorylation at the resting tone. The expression of MHC isoforms that differ in the C-terminus (SM1 and SM2) and in the N-terminal region (SM-A and SM-B), formed by alternative splicing of the pre-mRNA at either the 3' end or the 5' end, respectively, was analyzed. The expression of these isoforms was characterized at the mRNA and protein levels using reverse transcriptase-polymerase chain reaction (RT-PCR), SDS-PAGE, and Western blotting. The levels of MLC phosphorylation were 35.5 +/- 4.6, 24.7 +/- 2.2, 13.6 +/- 2.1, and 12.8 +/- 2.7 for dome, mid bladder body, base and urethra respectively. Almost 100% of the MHC mRNA in the dome, mid bladder body, and base contains a 7-amino acid insert near the ATP-binding region, whereas the MHC in the urethral smooth muscle is only 81% inserted. Prior studies have shown that inserted myosin has a two-fold higher actin-activated ATPase activity compared to the myosin isoform that lacks the insert, and the maximum velocity of shortening of smooth muscle containing this insert is high compared to muscle that do not contain the insert. The expression of SM1 and SM2 were not significantly different. Our data suggests the presence of a high degree of inserted myosin and LC20 phosphorylation in the bladder dome and mid-body helps to facilitate rapid force development and emptying. Non-inserted myosin and the low level of MLC phosphorylation in the urethra may contribute to slowly or non-cycling myosin cross bridges and the maintenance of a tonic or contracted state during bladder filling.

Pro-inflammatory cytokines induce expression of matrix-metabolizing enzymes in human cervical smooth muscle cells.

The process of cervical ripening has been likened to an inflammatory reaction associated with the catabolism of cervical extracellular matrix by enzymes released from infiltrating leukocytes. We hypothesized that smooth muscle cells in the cervix also participate in this process and that pro-inflammatory cytokines act on cervical smooth muscle cells (CSMC) to provoke the expression of matrix-degrading enzymes. We treated primary cultures of human CSMC with tumor necrosis factor-alpha (TNF-alpha) and examined expression of the elastinolytic enzyme, cathepsin S, the collagen metabolizing matrix metalloproteinases (MMP)-1, -3, -9, and the tissue inhibitor of metalloproteinase (TIMP)-1 and -2. A time course analysis revealed that 10 ng/ml of TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression with the maximal response observed after 24-48 hours. TNF-alpha induced cathepsin S, MMP-1, -3, and -9 mRNA expression in a dose-dependent manner: the maximal effect was observed at a concentration of 10 ng/ml, with appreciable increases observed at concentrations of 0.1 to 1.0 ng/ml. In contrast, TIMP-1 and -2 mRNAs were not significantly increased by TNF-alpha treatment. Interleukin-1beta produced a pattern of gene expression in the CSMC similar to that observed following TNF-alpha treatment. Western blot analysis and zymography confirmed the induction of proMMP-1, -3, and -9 in response to TNF-alpha, but MMP-2 immunoreactivity and zymographic activity were unaffected. TNF-alpha increased secretion of procathepsin S, but did not affect TIMP-1 and reduced TIMP-2 production. We conclude that CSMC are targets of pro-inflammatory cytokines, which induce a repertoire of enzymes capable of degrading the cervical extracellular matrix. The induction of these enzymes may facilitate the normal ripening of the cervix at term and participate in the premature cervical changes associated with preterm labor.

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis.

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries.

We demonstrate, using reverse transcriptase-polymerase chain reaction, that, whereas abdominal aorta from rabbit consists almost entirely of myosin heavy chain (MHC) mRNA with no insert at the 5'-terminal coding region, the distributing arteries (femoral and saphenous) begin to show MHC mRNA with the 21-nucleotide insert that encodes seven amino acids in the ATP-binding region located in the myosin head. The femoral/iliac artery contains > 50% inserted mRNA, whereas the more distal saphenous artery contains > 80% inserted mRNA. This insert is also present in the smooth muscle from rat tail artery but is absent in the smooth muscle from rat aorta. The actin-activated ATPase activity of myosin from the rabbit femoral/saphenous artery is 1.7-fold higher than that of the myosin from the aorta. A concomitant increase (about twofold) in the maximum shortening velocity of the saphenous artery, compared with that of the aorta, indicates that the preponderance of the inserted myosin is associated with both an increase in the actin-activated ATPase activity and a larger maximum velocity of shortening. Furthermore, analysis of the 17-kDa essential light chain from the aorta reveals near equal quantities of the 17-kDa light chain isoforms a and b, whereas the myosin from the femoral/ saphenous artery contains predominantly the 17-kDa light chain a isoform. Together, these data indicate that the smooth muscle cells from the small distributing arteries are similar to those of visceral smooth muscle with respect to the expression of myosin isoforms, actin-activated myosin ATPase activity and contractility.

Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells.

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4 degrees C) of the large molecular weight PDE4 denoted "Peak 2 PDE4" with mixed phosphatidic acids (PAs) produced a 2-fold increase in its Vmax without changing its Km (approximately 2 microM) for cyclic AMP. This "activation" was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.

Rolipram inhibition of phosphodiesterase-4 activation.

Rolipram inhibited U937 cell phosphodiesterase-4 in either the presence or absence of saturating (100 micrograms/ml) phosphatidic acid in an apparently phospholipid-independent manner, exhibiting similar kinetics (Ki values = 0.41 and 0.59 microM, respectively). At low concentrations (10 and 100 nM), however, rolipram caused a rightward shift of the phosphatidic acid concentration-response curve for phosphodiesterase-4 activation, suppressing activation by up to 70%. Maximum inhibition of phosphodiesterase-4 activation occurred at phosphatidic acid concentrations of 5-40 micrograms/ml. The results suggest that rolipram is capable of inhibiting phosphodiesterase-4 by both phospholipid-dependent and phospholipid-independent mechanisms.

Identification and stabilization of large molecular weight PDE-IVs from U937 cells.

Cytosolic cyclic nucleotide phosphodiesterases (PDEs) from human (promonocytic) U937 cells were rapidly resolved by DEAE-Sepharose CL-6B anion exchange chromatography into two major peaks of cAMP-specific activity possessing average Kms of 1.70 microM (Peak 1) and 1.65 microM (Peak 2). Both peaks were predominantly PDE-IV, but possessed molecular weights higher than those generally reported for partially purified PDE-IVs. Storage of Peak 2 for 24 h at 4 degrees C resulted in a doubling of its Vmax and an apparent decrease in its molecular weight. Activation of Peak 2 PDE-IV was prevented when the sodium acetate concentration in its buffer was reduced by dilution immediately following isolation. Although the relevance of this activation to cellular regulation of PDE-IV is undefined, the isolation and stabilization of PDE-IV in its large molecular weight form will be critical to future investigations of PDE-IV regulation.

A rapid method for detection of Yersinia enterocolitica serotype O:3 in pig feces using monoclonal antibodies.

A simple colony immunoblotting method using monoclonal antibodies (MAbs) was developed to detect Y. enterocolitica serotype O:3 in pig feces. One of the MAbs studied was able to detect single colonies of the organism in the presence of calculated 3.1 x 10(8) heterologous organisms in pig feces. The MAb was found to be specific for the lipopolysaccharide (LPS) O-antigens of Y. enterocolitica serotype O:3. No significant cross-reactivity was found against a variety of closely related serotypes and Gram-negative organisms. The MAb could also be used in a slide agglutination test and an indirect fluorescence antibody assay for rapid identification of Y. enterocolitica serotype O:3.

Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus.

A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.