PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N -benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N -benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.     

Body composition and energetic efficiency in two lines of mice selected for rapid growth rate and their F1 crosses

Summary Correlated responses to selection for increased growth rate were compared in two mouse populations (M16 and H₆) of distinct genetic origin. Traits studied were body composition, feed intake, constituent gains and energetic efficiency. When compared with their respective controls (ICR and C₂) at 6 and 9 weeks of age, body weight increased more in M16 (57%and 69 % of the control mean) than in H₆ (40 % and 34%). The M16 showed correlated responses in fat percent of 2.6% (P <.05), 8.4% (P <.01) and 11.2% (P <.01) at 3, 6 and 9 weeks, respectively, whereas corresponding values in H₆ were –2.4% (P <.05), 3.3% (P <.05) and 2.09 % (P >.05). The correlated responses in fat percent were 2.7 and 4.7 times higher in M16 than H₆ at 6 and 9 weeks. The regression of ln fat weight on ln empty body weight was larger in M16 (P <.05) compared to ICR and larger (P <.01) in H₆ compared to C₂. Both M16 and H₈ exhibited positive correlated responses from 3 to 6 weeks of age in feed intake and gain and efficiency in fat, protein, calories and ash; fat and caloric gain and efficiency exhibited higher correlated responses in M16 than H₆. During the 6- to 9-week interval, the M16 population continued to evince positive correlated responses in gains and efficiencies of fat, protein and calories, whereas H₆ did not. Several possible explanations are presented to account for the differences in correlated responses between the selected populations. Partitioning of correlated response differences between M16 and H₆ into average direct and average maternal genetic effects indicated that average direct genetic effects, favoring M16, were responsible for the major difference between the selected populations. Direct heterosis in F₁ crosses of the selected populations were generally not significant, although there was a tendency for fat percent and fat weight to show heterosis.Paper No. 4929 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27607, Animal Research Institute Contribution No. 624 and Agricultural University at Wageningen Contribution No. 654-490-10. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the Products named, nor criticism of similar ones not mentioned.On leave from Department of Animal Husbandry, Agricultural University, Wageningen, The Netherlands.On leave from Animal Research Institute, Agriculture Canada, Ottawa, Ontario K1A OC6.     

Direct and maternal genetic effects on body weight maturing patterns in mice

Summary Direct and maternal genetic effects were evaluated for maturing patterns of body weight in mice using a crossfostering design. Crossfostering was performed in one group using dams from populations selected for rapid growth rate (M16 and H₆) and their reciprocal F₁. crosses. A second crossfostering group consisted of dams from the respective control populations (ICR and C₂) and their reciprocal F₁. 's. Population differences were partitioned into direct and maternal effects due to genetic origin, correlated selection responses, heterosis and cytoplasmic or sex-linked effects. Degree of maturity was calculated at birth, 12, 21, 31 and 42 days of age by dividing body weight at each age by 63-day weight. Absolute and relative maturing rates were calculated in adjacent age intervals between birth and 63 days. Genetic origin effects (ICR vs. C₂; M16 vs. H₆) were significant for many maturity traits, with average direct being more important than average maternal genetic effects. In general, correlated responses to selection for maturity traits were larger in the M16 population (M16 vs. ICR) than in the H₆ population (H₆ vs. C₂) and correlated responses in average direct effects were larger than average maternal effects. Positive correlated responses in average direct effects were found for relative maturing rates at all ages and for absolute maturing rates from 31 to 63 days. Apparent correlated responses in degree of maturity were negative for M16 and H₆. However, further analysis suggested that the correlated response for degree of maturity in H₆ may be positive at later ages and negative at earlier ages. Direct and maternal heterosis for degree of maturity was positive in the selected and control crosses. Absolute and relative maturing rates showed positive heterosis initially, followed by negative heterosis. Reciprocal differences due to the cytoplasm or sex-linkage were not important for patterns of maturity.Paper No. 5244 the Journal Series of the North Carolina Agricultural Experiment Station, Ealeigh, Animal Research Institute Contribution No. 683 and Agricultural University at Wageningen Contribution No. 654–490–12On leave from the Animal Research Institute, Agriculture Canada at Ottawa, OntarioOn leave from the Department of Animal Husbandry, Agricultural University at Wagenitgen, the Netherlands     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

Comparative plankton investigations in a brackish lake (L. Veere) and a brackish tidal estuary (Westerschelde) in the SW Netherlands

Summary As the data, presented in this lecture, have been published elsewhere (see references), only a summary is given here.A comparison was made between the plankton assemblages of the mesoand polyhaline ranges of Lake Veere and the Westerschelde estuary. Species composition, densities and seasonal distributions of the predominant planktonic organisms of the investigated brackish water ecosystems were quite different, depending on several other environmental factors than salinity.     

N-Acetylhistidine, Glutamate, and β-Alanine Are Concentrated in a Receptor Cell Layer of the Trout Inner Ear

An epithelial sheet isolated from the trout saccular macula, highly enriched in acousticolateralis receptor cells (hair cells), has been analyzed for primary amine-containing compounds. The hair cell preparation, compared to the saccular nerve, was found to contain elevated levels of the presumptive receptoneural transmitter, glutamate, as well as beta-alanine, and components eluting in the positions of the standards phosphoserine and phosphoethanolamine on cation-exchange HPLC. Saccular nerve contained a different spectrum of primary amines and was elevated specifically in carnosine/homocarnosine. Acid hydrolysis of perchlorate extracts of both hair cell and nerve fractions yielded large amounts of histidine. For the saccular nerve fraction, production of histidine by acid hydrolysis was matched by production of beta-alanine and gamma-aminobutyric acid (GABA) and disappearance of carnosine/homocarnosine. The dipeptides carnosine and homocarnosine have been chromatographically resolved by expanded HPLC and found to be present in saccular nerve in a ratio of approximately 10:1, respectively. Production of histidine in the hair cell extract was not coupled with production of beta-alanine and GABA. The hair cell histidine-containing unknown, present in millimolar concentration, has been identified as N-acetylhistidine by the hydrolysis and rechromatography of fractions from cation-exchange HPLC. The large and specific presence of N-acetylhistidine in the hair cell preparation, together with electrophysiological evidence for its facilitatory action on afferent fibers in the frog semicircular canal, is suggestive of a role for this molecule as well as glutamate in acousticolateralis receptoneural transmission.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.