Cystine Aminopeptidase Research Articles

Systemic Arterial Vasodilation, Vasopressin, and Vasopressinase in Pregnancy

Systemic arterial vasodilation in early pregnancy is accompanied by a compensatory rise in cardiac output and a decline in BP. This relative arterial underfilling in early pregnancy is coupled to stimulation of the renin-angiotensin-aldosterone system and hypotonicity. Arterial underfilling induces the nonosmotic stimulation of arginine vasopressin and upregulation of aquaporin 2 followed by trafficking of this water channel to the apical membrane of principal cells along the collecting ducts. In middle and late pregnancy, there also is a four-fold increase in vasopressinase, a cystine aminopeptidase produced by placental trophoblasts, which enhances the metabolic clearance of vasopressin. In the setting of preeclampsia, twins or triplets, or subclinical central diabetes insipidus, a transient diabetes insipidus may ensue from this vasopressinase-mediated degradation of N-terminal amino acids from the vasopressin molecule. Because desmopressin is already deaminated at the N-terminal, it is resistant to the effect of vasopressinase and therefore is the treatment of choice for transient diabetes insipidus of pregnancy.

Diabetes insipidus in pregnancy.

Diabetes insipidus is an uncommon condition with various aetiologies. Recent research has uncovered new mechanisms underlying the syndrome. Careful attention to management is essential in pregnant women to avoid serious complications. Diabetes insipidus in pregnancy may be due to relative reduction in secretion of AVP from the posterior pituitary (cranial DI), increase in breakdown of AVP by placental cystine aminopeptidase with vasopressinase activity, or resistance of the rental tubules to AVP (nephrogenic DI).

Biochemical composition and digestive enzyme activity during naupliar development of Artemia spp from three solar saltworks in Greece

Three parthenogenetic brine shrimp Artemia populations from saltworks in Greece (Messolongi, Milos and Polychnitos) were studied with regard to their biochemical composition and the activity of digestive enzymes in four developmental stages from decapsulated cysts to nauplii instar III. The content of protein and RΝΑ was highest in nauplii instar II (65.10 and 6.23% dry weight, respectively), while that of total lipid was highest in nauplii instar I (11.40% dw). The carbohydrate content was highest in decapsulated cysts (10.30% dw) and about one half that value in nauplii instar I. The DNA content was ten-fold higher in nauplii instar I than in decapsulated cysts with the highest levels in nauplii instar III (3.67% dw). The activity of alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, β-galactosidase, β-glucosidase, esterase lipase (C8), esterase (C4), N-acetyl-β-glucosaminidase, α-fucosidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase increased significantly from decapsulated cysts to nauplii instar II and remained constant or decreased in nauplii instar III. In nauplii instar II, leucine aminopeptidase, β-galactosidase, alkaline phosphatase and esterase (C4) demonstrated higher activity levels (140.90, 133.62, 66.01 and 45.47 nmoles/100 µg dw within 5 min, respectively) of the aforementioned enzymes. The Artemia population of Messolongi had significantly higher levels of DNA, protein and alkaline phosphatase activity per individual, as well as body weight, compared to the Artemia populations of Milos and Polychnitos. The latter population showed the lowest level of lipid and the highest esterase activity (C4) per individual. There was a reverse relationship between the protein content and leucine aminopeptidase activity, lipid content and esterase (C4) activity, carbohydrate content and β-galactosidase activity among the three Artemia populations. The variations observed in the biochemical composition and the activity of some specific enzymes should be taken into consideration in selecting these Artemia populations from Greece for application in aquaculture.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: II. Establishment and maintenance of the uterine muscular twisting, a strategy for long-term sperm storage

Crotaline snakes store sperm by means of a uterine musculature twisting (UMT). We investigated the influence of plasma levels of estradiol and progesterone and vasotocinase cystine aminopeptidase (CAP) activity on UMT formation and maintenance, and the in vitro uterine reactivity for AVT in Crotalus durissus terrificus in primary or secondary vitellogenesis with or without UMT. Frequency of females in secondary vitellogenesis with UMT is significantly higher than in primary one. Estradiol levels did not vary in all conditions studied, however, significantly low levels of progesterone were found in snakes in secondary vitellogenesis with UMT compared to those without it. UMT is always observed when high levels of estradiol and low levels of progesterone are detected. CAP activity did not change in the presence of UMT. AVT produced concentration–response contractions of the isolated uterus of snakes in all stages analysed and the pD 2 value and maximum contractile response were significantly higher in primary vitellogenesis without UMT than in other reproductive conditions, indicating that uterus of those snakes presents a higher contractile capacity which may favour UMT establishment. In conclusion, we show a relationship of UMT and estradiol/progesterone balance and a possible participation of AVT in UMT formation and maintenance in the Neotropical rattlesnake.

Reproductive cycle of the Neotropical Crotalus durissus terrificus: I. Seasonal levels and interplay between steroid hormones and vasotocinase

Crotaline snakes present delayed fertilization and sperm storage because secondary vitellogenesis is not completed by the time of mating. The release of vitellogenesis and synchrony between ovulation and fertilization suggest a steroidal modulation. We investigated changes of sexual steroid levels during reproduction in the Neotropical rattlesnake Crotalus durissus terrificus, analyzing macroscopical variations of reproductive condition (vitellogenesis, pregnancy, and post-partum) and plasma levels of estradiol, progesterone, and vasotocinase cystine aminopeptidase (CAP) activity over 2 years. Data showed 44.4% non-reproductive snakes (40.1% primary vitellogenesis and 4.3% post-partum) and 55.6% reproductive (36.8% secondary vitellogenesis and 18.8% pregnant). Estradiol was low in spring and summer, increasing in autumn till it peaked in winter. Estradiol in secondary vitellogenesis was significantly higher than in primary vitellogenesis, or in pregnant and post-partum females, Progesterone dropped significantly in autumn compared to summer, winter, and spring. Pregnant females showed the highest levels of progesterone compared to primary or secondary vitellogenesis, or post-partum females. CAP activity showed lowest values in reproductive females in autumn and greatest levels in post-partum females. A significant negative linear relationship was obtained between CAP activity and estradiol. The combination of morphological observations, levels of steroids and CAP activity allowed us to suggest a similar morphological reproductive pattern between temperate and tropical rattlesnakes, and to infer the role of estradiol, progesterone and CAP activity on vitellogenesis, gestation and sperm storage, respectively.

Tissue distribution of placental leucine aminopeptidase/oxytocinase during mouse pregnancy.

Placental leucine aminopeptidase (P-LAP), also called oxytocinase, is an enzyme responsible for hydrolyzing oxytocin. This enzyme is identical to cystine aminopeptidase. We examined the tissue distribution of P-LAP in normal adult mice and also in mothers and fetuses during mouse pregnancy using immunohistochemical (IHC) analysis. P-LAP-immunoreactive protein was expressed in various organs in a cell- and gestational stage-dependent manner. In the kidney, P-LAP was located in distal and collecting tubules but not in proximal tubules. The islet of Langerhans in the maternal pancreas stained positively for P-LAP in the periphery in early gestation. This staining pattern changed so that both the periphery and inner cells were positive during mid-gestation and finally only inner cells were positive in late gestation. Among the hematopoietic cells in the fetal liver, only megakaryocytes showed strong expression of P-LAP. The staining intensity increased with gestation on the apical surface of trophoblasts in the placental labyrinth. These data demonstrate that P-LAP is present in a variety of tissues, and its presence is affected by pregnancy and fetal development. Therefore, P-LAP may play a significant role in various physiological processes in non-pregnant, pregnant, and fetal mice.

Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells

Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10 −7 M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10 −7 M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.

Interleukin-1beta stimulates placental leucine aminopeptidase/oxytocinase expression in BeWo choriocarcinoma cells.

In addition to prostaglandins, inflammatory cytokines induce uterine contraction via oxytocin (OT). Placental leucine aminopeptidase (P-LAP), an oxytocinase that is identical to cystine aminopeptidase, destroys OT activity. Patients with spontaneous preterm delivery have higher concentrations of inflammatory cytokines and lower P-LAP activities than those with normal delivery. In addition, the P-LAP promoter region contains putative binding sites for cytokine-induced transcription factors. We therefore postulated that inflammatory cytokines suppress P-LAP expression and examined this notion using BeWo choriocarcinoma cells cultured in the presence of cytokines. However, interleukin-1beta (IL-1beta) increased P-LAP activity in a time- and dose-dependent manner. Furthermore, Western blot analysis showed a dose-dependent increase of P-LAP proteins. We also detected IL-1 type I receptor mRNA in BeWo cells by RT-PCR. Semi-quantitative RT-PCR and Southern blot analysis showed that IL-1beta also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1beta-induced P-LAP mRNA accumulation within 1.1 kb upstream of the P-LAP gene. Immunohistochemical analysis of human placenta with chorioamnionitis demonstrated prominent P-LAP staining at sites of abundant inflammatory cell infiltration. These findings indicated that prolonged exposure to IL-1beta induces P-LAP in the trophoblasts, possibly via other de-novo protein synthesis, which contradicted our initial hypothesis.

Microbial Population Dynamics and Enzyme Activities During Composting

The changes in population size of different microbial groups (total aerobic heterotrophs, actinomycetes, fungi, fecal coliforms, ammonium- and nitrite-oxidizing bacteria, and denitrifying bacteria) and the activities of 19 different enzymes (three phosphatases, three esterases, two proteases, three amino-peptidases, and eight glycosyl-hydrolases) were examined during cocomposting of poultry litter (a mixture of poultry manure, waste feed, feathers, and wood shavings) and yard trimmings (a mixture of grass clippings, leaves, and wood barks). Three piles with forced aeration were established by mixing 2:1 (v/v) ratio of poultry litter and yard trimmings. During composting, samples were taken at three different locations (top, middle, and bottom) of the forced aeration piles for microbial and enzyme analyses. Results demonstrated that population size of different microbial groups was not a limiting factor in this composting process as the microorganisms in the poultry litter + yard trimmings compost are in great abundance. Although the numbers of these microbial groups were reduced by high temperature, their populations multiplied rapidly as composting progressed. Fecal coliforms were eliminated by day 49, suggesting that the poultry litter + yard trimmings compost showed an overall increase in diversity and relative abundance of extracellular enzymes present as composting progressed. The population of fungi and actinomycetes (microorganisms active in degradation of cellulose, hemicellulose, and lignin) were positively correlated with esterase, valine amino-peptidase, α-galactosidase, β-glucosidase, and lipase. Of all 19 enzymes examined, ß-galactosidase (enzyme involved in the hydrolysis of lactose) had the most significant positive correlation with microbial populations, such as total aerobic heterotrophs, ammonium- and nitrite-oxidizing bacteria, denitrifying bacteria, and fecal coliforms. Cystine amino peptidase, chymotrypsin, and trypsin showed no evidence of activity during the entire period of composting. This composting process represented a combined activity of a wide succession of environments in the compost pile as one microbial group/enzyme overlapped the other and each emerged gradually as a result of the continual change in temperature as well as moisture content, O2 and CO2 level, and progressive breakdown of complex compounds to simpler ones.

AP-2 and Ikaros Regulate Transcription of Human Placental Leucine Aminopeptidase/Oxytocinase Gene

P-LAP is identical with cystine aminopeptidase as oxytocinase. We previously located on the placental leucine aminopeptidase (P-LAP) gene the footprint site with the high promoter activity (FP3: −214 to −183) and found a possible interaction between it and AP-2 in choriocarcinoma cells. Here, we investigated FP3 in detail and identified the elements responsible for the high basal rate of transcription. Electrophoretic mobility shift assays demonstrated that FP3 would interact with Ikaros as well as AP-2. Further analysis using antibody against Ikaros confirmed that Ikaros was indeed present and bound to FP3. In addition, AP-2α and AP-2γ antibodies supershifted the second complex at FP3. Functionally, mutations that eliminate AP-2 binding reduced promoter activity significantly, while those that eliminate Ikaros binding reduced promoter activity insignificantly. Double mutations of AP-2 and Ikaros decreased promoter activity progressively. We conclude that AP-2 is the main activator and Ikaros functions cooperatively with it for maximal expression of the human P-LAP gene.

Interactions among challenges of hydromineral balance, angiotensin-converting enzyme, and cystine aminopeptidase

Enzymatic cleavage of some peptides could be included among the mechanisms of water-electrolyte homeostasis. To test this hypothesis, the angiotensin-converting activity (ACE) of plasma and the L-cystine-di-β-naphthylamidase activity (CAP) of plasma and of soluble and particulate fractions from different areas of the central nervous system (CNS) were investigated in rats submitted to treatments eliciting hydromineral imbalance. CAP in the CNS was unchanged by hydromineral challenges. The correlations observed between plasma osmolality and CAP, and plasma CAP and ACE suggested a contribution of these activities to the restoration of basal water-electrolyte and blood pressure conditions through the hydrolysis of vasopressin, oxytocin, angiotensin I and bradykinin.

Extracellular enzyme profiles during co-composting of poultry manure and yard trimmings

APYZYM™ assays were used to detect the presence of enzyme activity from 19 different enzymes, including three phosphatases, three esterases, three amino-peptidases, and eight glycosyl-hydrolases during co-composting of poultry manure and yard trimmings (poultry+yard trimmings). Results of this study have shown that the poultry+yard trimmings compost went through physico-chemical and biochemical changes during composting. These changes included self-heating of the compost mass, relative increases in enzyme activities, decreases in water-soluble components (i.e. water-extractable C, inorganic N, and heavy metal contents), and elimination of phytotoxicity. An overall increase in enzyme activity was observed over the course of the present experiment. Alkaline and acid phosphatase, and leucine amino-peptidase activities were high, while lipase, esterase, and esterase activities were moderate at the beginning of composting. Cystine amino peptidase, chymotrypsin, and trypsin showed no evidence of activity during the entire period of composting. Of the eight glycosyl-hydrolases, only α-galactosidase, β-glucosidase, and N-acetyl-β glucosaminidase showed any significant activity, fluctuating between low and moderate activity. The activity of α-mannosidase was low while β-galactosidase, β-glucuronidase, and α-fucosidase remained undetected during the entire testing period). Although this enzyme test is rather preliminary, the results of this study seem to show potential usefulness of enzyme activity measurements as indices of the course of the actual composting.

Placental leucine aminopeptidase/oxytocinase in maternal serum and placenta during normal pregnancy

Placental leucine aminopeptidase (P-LAP), which is identical with cystine aminopeptidase as oxytocinase, was found to be homologous with rat insulin-regulated membrane aminopeptidase (IRAP) by sequence comparison. In the current study, we determined the P-LAP levels in maternal serum and placenta during healthy pregnancy. P-LAP activities in maternal serum increased with gestation and rose to the peak of 80 IU/ml at 38 weeks of gestation. Northern blot analysis revealed the increase of P-LAP mRNA levels in placenta in the third trimester compared to the first trimester. P-LAP protein and related activities could be detected in the conditioned medium of placental tissue, while they could not be detecetd in that of human umbilical vein endothelial cells. Immunohistochemically P-LAP was positively stained in the apical membrane of syncytiotrophoblast cells throughout the gestation. These results established the normal range of serum and tissue P-LAP levels during pregnancy and the possible source of serum P-LAP, which will be helpful to elucidate the physiological and clinical roles of P-LAP/oxytocinase/IRAP.

Structural Organization of the 5′-End and Chromosomal Assignment of Human Placental Leucine Aminopeptidase/Insulin-Regulated Membrane Aminopeptidase Gene

Placental leucine aminopeptidase (P-LAP) which is identical with cystine aminopeptidase as oxytocinase was found to be a homologue of rat insulin-regulated membrane aminopeptidase (IRAP) by cDNA cloning. In this study, we confirmed 5′-end cDNA sequence of P-LAP and isolated genomic clones containing the upstream region of human P-LAP gene. The transcription initiation sites determined by primer extension located 478 and 480 bp upstream of the initiation methionine codon, 38 bp downstream of TATA box-like motif. The 5′-flanking region of human P-LAP gene contained DNA-binding motifs for several ubiquitous transcription factors such as SP1 and AP2. Chromosomal localization by fluorescence in situ hybridization showed that the gene was assigned to 5q14.2-q15 of the human chromosome. This study establishes the genetic basis for P-LAP gene research, thereby leading to better understanding of the molecular mechanism underlying the P-LAP gene.

Changes in cystine aminopeptidase (oxytocinase) activity in mouse serum, placenta, uterus and liver during pregnancy or after steroid hormone treatments.

Changes in cystine aminopeptidase (CAP) activity in serum, placenta, uterus and liver in mice under various physiological conditions were examined by enzymatic analysis using S-benzyl-L-cysteine-4-methylcoumaryl-7-amide as a substrate. Changes in serum CAP activity during pregnancy were less remarkable than those reported in humans; the activity was highest at day 13 of pregnancy and lowest at day 17. During the estrous cycle, the serum CAP level was high at diestrus, as high as that at day 13 of pregnancy. The activity in the male serum was significantly low compared to that in the diestrous serum. Although the CAP level was especially high in the maternal placenta, levels were also high in the fetal placenta, uterus and liver as compared with the serum, suggesting CAP synthesis in these tissues. The CAP activity in the uterine tissue was lower in pregnant than normal cycling mice. Furthermore, the serum CAP activity was modulated by estrogen and progesterone both in females and males, and by androgen in males. The relevance of these findings to the physiological role of CAP was discussed.

Osmoregulation, the secretion of arginine vasopressin and its metabolism during pregnancy

This review stresses changes in osmoregulation as well as the secretion and metabolism of arginine vasopressin during pregnancy, focusing on human gestation. Pregnant women experience a decrease in body tonicity, plasma osmolality decreasing immediately after conception to a nadir approximately 10 mosmol/kg below non-pregnant levels early in pregnancy, after which a new steady state is maintained until term. Data from both human and rodent gestation have led to a formation of how these changes occur. The osmotic thresholds for thirst and antidiuretic hormone release decrease in parallel. Lowering the threshold to drink stimulates increased water intake and dilution of body fluids. Because arginine vasopressin (AVP) release is not suppressed at the usual level of body tonicity, the hormone continues to circulate and the ingested water is retained. Plasma osmolality declines until it is below the osmotic thirst threshold, and a new steady state with little change in water turnover is established. Pregnancy is characterized by increments in intravascular volume, but volume-sensing AVP release mechanisms appear to adjust as gestation progresses so that each new volume status is "sensed" as normal. The metabolic clearance of AVP increases fourfold, the rise paralleling that of circulating cystine aminopeptidase (vasopressinase), and enzyme produced by the placenta. Furthermore, the disposal rate of 1-deamino-8-D-AVP, and AVP analogue resistant to inactivation by vasopressinase, is unaltered in pregnancy. Thus, the increase in AVP's metabolism and the high circulating aminopeptidase levels have been implicated in certain forms of transient diabetes insipidus that occur in late pregnancy. Finally, mechanisms responsible for the altered osmoregulation in pregnancy are obscure, but chorionic gonadotropin and relaxin may be implicated in the changes.

Identification of an alanine aminopeptidase in human maternal serum as a membrane-bound aminopeptidase N.

In addition to cystine aminopeptidase (oxytocinase) alanine aminopeptidase is present at high levels in the serum of pregnant women. In this study we compared the enzyme with membrane-bound aminopeptidase N purified from human placenta. Comparison of catalytic and immunological properties and N-terminal sequence analyses revealed that the enzymes were differentially processed derivatives of the same protein, and that the N-terminal 68 residues of aminopeptidase N were deleted in the alanine aminopeptidase. The deleted sequence contains a small cytoplasmic region, a hydrophobic transmembrane domain and a junctional domain. These results suggest that the enzyme may be released into the maternal circulation as a result of lacking these three domains.

Elution profiles and molecular weights of placental cystine aminopeptidase in animals.

Gel filtration was performed for cystine aminopeptidase (CAP) [EC 3.4.11.3] on full term placental extracts from human, cynomolgus monkey, dog, goat, pig and horse. The enzymatic profiles of CAP were examined and compared with that of CAP in pregnancy plasma on the basis of inhibitory effects. Elution profiles of placental extracts exhibited 2 CAP activity peaks in human and pig, 3 peaks in cynomolgus monkey, dog and goat, and 4 peaks in horse. The molecular weights of placental CAP that showed identical inhibitory effects to that of pregnancy plasma CAP were estimated to be approximately 325,000 in human, 350,000 in the cynomolgus monkey, 140,000 in the dog, 140,000 in the goat, 128,000 in the pig, and 115,000 in the horse. These molecular weights tended to decrease in accordance with the increase of barrier layers present between maternal blood and placental syncytiotrophoblasts in which CAP is synthesized.

The effect of early labour, maternal analgesia and fetal acidosis on fetal plasma oxytocin concentrations.

To determine the effect of early labour, maternal analgesia and fetal hypoxia on circulating fetal oxytocin concentrations. Prospective observational study. Delivery suite in a District General Hospital. Fifty women at term who did not require oxytocin administration or more than one form of analgesia. Study groups: vaginal delivery with (1) no analgesia, (2) pethidine, or (3) epidural analgesia. Caesarean section under regional analgesia (4) prior to, and (5) after the onset of labour. Samples of blood were collected from the umbilical artery (UA) and umbilical vein (UV) immediately after fetal delivery prior to placental separation or oxytocic administration. Plasma oxytocin (OT) concentration, umbilical vein pH, cystine aminopeptidase activity. The geometric mean UA-OT was significantly greater than UV-OT in all groups and was not altered by pethidine; however, epidural administration increased the UA-UV difference. The UA-UV difference at caesarean section was not significantly altered by the onset of labour. There was no correlation between UV pH and UA-UV plasma oxytocin. Cystine aminopeptidase activity was not detectable in UA and UV plasma. Fetal OT production is increased by epidural but not by pethidine analgesia. It is not influenced by the onset of labour or fetal hypoxia.

Identification of Pasteurella haemolytica A1 isolates from market-stressed feeder calves by use of enzyme and antimicrobial susceptibility profiles

SUMMARY An epidemiologic study of Pasteurella haemolytica serovar 1 (Ph1) in market-stressed feeder calves from 7 farms in eastern Tennessee was conducted. The nasal mucus of each calf was cultured sequentially at the farm of origin (day 0), at an auction market (day 133), and at a feedyard in Texas (days 141, 148, 155, and 169). Of the 103 calves tested, 77 were culture-positive, including 1 on day 0, 1 on day 133, 20 on day 141, 57 on day 148, 50 on day 155, and 14 on day 169. From the 143 Ph1 isolates, 20 enzyme profiles were determined by use of a commercial enzyme system that detects 19 enzymatic reactions; 4 antimicrobial susceptibility profiles were obtained, using the disk-diffusion method, which evaluated susceptibility to 11 antibacterial drugs. All isolates were positive for acid phosphatase and alkaline phosphatase, but were negative for α-galactosidase, α-mannosidase, β-glucosidase, β-glucuronidase, cystine aminopeptidase, N-acetyl-β-glucosaminidase, and trypsin. Other positive enzyme reactions included: leucine aminopeptidase, 140 Ph1 isolates; phosphohydrolase, 90 isolates; α-fucosidase, 63 isolates; esterase (C4), 59 isolates; valine aminopeptidase, 30 isolates; esterase lipase (C8), 24 isolates; β-galactosidase, 2 isolates; and α-glucosidase, chymotrypsin and lipase (C14), 1 isolate each. Thirty-four Ph1 profiles were identified, using combined enzyme and antimicrobial susceptibility profiles. The data indicate that the strains isolated during the feedyard period may have been determined more by farm of origin (P ≤ 0.001) than by habitation with calves from other farms while in the feedyard. The combined enzyme and antimicrobial susceptibility profile method is a rapid and simple epidemiologic technique for tracking Ph1 strains in market-stressed feeder calves.