Gene dosage effect for fumarate hydratase (FH; E.C. 4.2.1.2) in partial trisomy 1

Summary A strain of fibroblasts partially trisomic for the larger part of 1q (Norwood and Hoehn, 1974) contains about 1.5 times as much fumarate hydratase (FH) as various control-strains. This gene dosage effect was ascertained by (1) comparative measurements of the specific activity; (2) relating the specific activity of FH to that of reference enzymes, not influenced by the chromosomal anomaly; and (3) by immunoprecipitation methods, using a rabbit antiserum against pig heart FH which cross-reacts with the human enzyme. Among others, this gene dosage effect can be demonstrated numerically by the following parameters:Ratio of the average specific activity of FH in the trisomic strain to that of the control strains: 1.53.Corresponding ratio after dividing FH activity by that of reference enzymes; for acid phosphatase: 1.58, for glutamate dehydrogenase: 1.53.Average ratio of the immunoprecipitation areas obtained upon radial immunodiffusion according to Mancini et al. (1965): 1.56.This paper is dedicated to the painter and philosopher Helmut Berninger on the occasion of his 50th birthday     

Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E.

The only dipeptide found to serve as a leucine source for a Salmonella strain lacking peptidases N, A, B, D, P, and Q was alpha-L-aspartyl-L-leucine. A peptidase (peptidase E) that specifically hydrolyzes Asp-X peptides was identified and partially purified from cell extracts. The enzyme (molecular weight, 35,000) is inactive toward dipeptides with N-terminal asparagine or glutamic acid. Mutants (pepE) lacking this enzyme were isolated by screening extracts for loss of the activity. Genetic mapping placed the pepE locus at 91.5 map units and established the gene order metA pepE zja-861::Tn5 malB. Duplications of the pepE locus showed a gene dosage effect on levels of peptidase E, suggesting that pepE is the structural gene for this enzyme. Mutations in pepE resulted in the loss of the ability to grow on Asp-Pro as a proline source but did not affect utilization of other dipeptides with N-terminal aspartic acid. Loss of peptidase E did not cause a detectable impairment in protein degradation. Two other peptidases present in cell extracts of mutants lacking peptidases N, A, B, D, P, Q, and E also hydrolyze many Asp-X dipeptides.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

谷子雄性不育系1066A不育基因和黄苗基因的染色体定位

以谷子(Setaria italica (L.) Beauv.)雄性不育系1066A为母本,豫谷1号三体(1～7)及四体8和四体9作父本进行杂交,应用初级三体分析法,进行了谷子雄性不育基因和黄苗基因的染色体定位研究.通过配置大量杂交组合和反复授粉,利用豫谷1号三体的极少量花粉,获得了三体2～9的F1代杂种,各杂种三体的形态与豫谷1号三体基本相似,略有差异,苗色呈绿色且可育.杂种F2植株的苗色和育性都产生分离.结果是三体3、5、7、8、9的F2代分离出的可育株与不育株之比为3∶1,三体6的可育株与不育株之比为14∶1 (χ2=0.012,P=0.01).杂种F2分离出的绿苗与黄苗之比只有三体7为12∶1 (χ2=0.36, P=0.01),其他均为3∶1.因此,可以确定1066A的不育基因为隐性单基因,位于第6号染色体上,该品系的黄苗基因也是隐性单基因,位于第7号染色体上.     

Analysis of Autosomal Dosage Compensation Involving the Alcohol Dehydrogenase Locus in Drosophila Melanogaster

An example of autosomal dosage compensation involving the expression of the alcohol dehydrogenase (Adh) locus is described. Flies trisomic for a quarter of the length of the left arm of chromosome two, including Adh, have diploid levels of enzyme activity and alcohol dehydrogenase messenger RNA. Subdivision of the compensating trisomic into smaller ones revealed a region that exerts an inverse regulatory effect on alcohol dehydrogenase activity and messenger RNA levels and a smaller region surrounding the structural gene that exhibits a direct gene dosage response. The two opposing effects are of sufficient magnitude that they cancel when simultaneously present resulting in the observed compensation in the larger aneuploid. An Adh promoter-white structural gene fusion construct is affected by the inverse regulatory region indicating that the effect is mediated through the Adh promoter sequences. The role of autosomal dosage compensation in understanding aneuploid syndromes and karyotype evolution in Drosophila species is discussed.     

Is the peptidase activity of highly purified human plasma cholinesterase due to a specific cholinesterase isoenzyme or a contaminating dipeptidylaminopeptidase?

The peptidase site of human plasma cholinesterase (butyrylcholinesterase) is distinct from its esteratic site. We found that the number of peptidase sites on an enzyme highly purified from pooled plasma is less than 0.1, as compared with 4 esteratic sites, per tetramer. However, the subunits which carry the peptidase sites are electrophoretically indistinguishable from esteratic subunits. The atypical-silent enzyme (Ea1Es1) had a much higher absolute peptidase activity when substance P was used as the substrate, and we found that the number of peptidase and esteratic sites of this enzyme was roughly the same. This suggests that the mutated esteratic site of the silent possesses a peptidase activity. The esteratic site of the usual allozyme (Eu1Eu1) has no peptidase activity towards substance P. However, a small proportion of peptidase subunits are present in all preparations of enzymes purified from the plasmas of homozygote individuals. The peptidase activity of butyrylcholinesterase might therefore correspond to a specific isoenzyme produced by an epigenetic mechanism or produced by a gene distinct from genes E1 and E2 encoding for cholinesterase subunits. However, the possibility that highly purified cholinesterase contains traces of a dipeptidylaminopeptidase cannot be completely ruled out.     

Discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 by a single mutation

It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg(526)) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is approximately 7 times higher than the peptidase activity with Ac-Leu-p-nitroanilide as substrate. However, with the same substrates, this difference was increased to approximately 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on k(cat) and K(m) of the enzyme were discussed based on molecular dynamics simulation studies.     

Dosage effects for superoxide dismutase-1 in nucleated cells aneuploid for chromosome 21.

Assays of the activity of chromosome 21 determined superoxide dismutase-1 (SOD-1) in lymphocytes and polymorphonuclear granulocytes have demonstrated 38% and 40% increases, respectively, in cells from individuals with trisomy 21. Similarly, SOD-1 activity in trisomic fibroblasts is increased by 81%, while cells monosomic for chromosome 21 have only 60% of normal activity. Taken together with the data on SOD-1 activities in trisomic erythrocytes and platelets, the present results firmly confirm the existence of a true dosage effect for this enzyme in cells aneuploid for chromosome 21. However, the results of assays of the activity of glutathione peroxidase in trisomic fibroblasts did not confirm the possibility previously reported of a chromosome 21 related dosage effect for this enzyme.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Genetics and regulation of peptidase N in Escherichia coli K-12

Escherichia coli K-12 strains contain a cytoplasmic activity, peptidase N, capable of hydrolyzing alanine-p-nitroanilide. Mutations in the structural gene for the enzyme, pepN, were mapped, and the properties of mutant strains were examined. The pepN locus lay between ompF and asnS at approximately 20.8 min on the E. coli chromosome. Loss of peptidase N activity through mutation had no apparent effect on the growth rate or nutritional needs of the cell. Enzyme levels in wild-type strains were constant throughout the growth cycle and were constitutive in all of the growth media tested. Starvation for carbon, nitrogen, or phosphate also did not alter enzyme levels. Constitutive expression of peptidase N is consistent with the idea that the enzyme plays a significant role in the degradation of intracellularly generated peptides.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Genetic and Environmental Effects on the Expression of Peptidases and Larval Viability in Drosophila Melanogaster

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.     

Thermostabilization of carboxypeptidase A by interaction with its monoclonal antibodies.

The effect of interaction of carboxypeptidase A (CPA) with three monoclonal antibodies, each with a different epitope (CP 10, CP 9, and CP 8), on the heat stabilization of enzymes is described. These monoclonal antibodies bind to CPA with a relatively high binding constant (approximately 10(8) M-1) and do not affect its catalytic properties. Intact carboxypeptidase A lost more than 95 and 90% of its esterase and peptidase activities within 120 min at 50 degrees C. The monoclonal antibodies increased the thermal stability of the enzyme by 90 and 60%, as compared with the peptidase and esterase initial activities, respectively. Binding of these monoclonal antibodies, alone or in pairs, to the enzyme epitopes that are supposedly involved in heat denaturation of CPA result in stabilization of the conformation of the enzyme. The effect of thermostabilization by monoclonal antibodies was more pronounced with respect to peptidase activity than to esterase activity, indicating that these activities follow different reaction mechanisms. Since properly selected monoclonal antibodies can be prepared against virtually any enzyme, their immunocomplexation may provide a general and convenient method for stabilization of the enzyme conformation to heat denaturation, without affecting the catalytic properties.     

Purification and characterization of tripeptidyl peptidase I from Dictyostelium discoideum.

A tripeptidyl peptidase I from Dictyostelium discoideum was purified 744-fold to near homogeneity. The enzyme is 214 kDa in size and is composed of two monomers with a M(r) of 107 kDa. It has two pH optima at pH 4.5 and 5.9 and is a serine peptidase with no aminopeptidase or dipeptidyl peptidase activity. The enzyme was relatively specific showing activity on ala-ala-phe-p-nitroaniline but also acted on substrates with proline in the P1 position in contrast to mammalian TPP I. The K(m) values of the enzyme at pH 4.5 for ala-ala-phe-, ala-phe-pro- and ala-ala-pro-p-nitroanilines were 27 microM, 437 microM and 888 microM, respectively. The enzyme is most abundant during the amoeba stage of the life cycle but is present in the early stages of development and may therefore have a dual role in the organism in mobilizing amino acids or in processing specific peptides or proteins.     

Ultrastructural studies of the nucleoli in diploid and trisomic chickens

Chickens having a trisomic complement for the nucleolar organizer region (NOR) serve as a model for investigations on gene expression, molecular regulation, and physiological alterations resulting from extra genetic material. Of fundamental importance is the question of whether the extra set of ribosomal cistrons in trisomic chickens generates a morphologically and biochemically complete nucleolus. Ultrastructural and cytochemical studies of diploid and trisomic embryonic and adult cells were undertaken. All structures identified in trisomic cells as nucleoli by gross morphology were shown to be RNA rich by acridine orange fluorescence. At the ultrastructural level, the expected granular and fibrillar components were identified in all nucleoli of trisomic cells. Fibrillar centers, indicative of NOR's, were seen in all cases. These and other results from silver staining suggest full gene activity for each of the three chromosomes in the trisomic cell at all stages of development.     

Leukotriene A4 hydrolase: an anion activated peptidase.

The peptidase activity of leukotriene A4 hydrolase purified from human leukocytes has been characterized, utilizing synthetic amides as substrates. The enzyme was stimulated by several monovalent anions. Thiocyanate ions were most effective followed by chloride and bromide ions. In phosphate buffer alone the peptidase activity towards alanine-4-nitroanilide was barely detectable and addition of 100 mM NaCl increased the specific activity more than 20-fold. Increasing the concentration of NaCl (or NaSCN) did not significantly affect the apparent Km for the substrate alanine-4-nitroanilide, but resulted in a dose dependent increase of Vmax. The stimulatory effect of these anions on the reaction velocities appeared to obey saturation kinetics and thus indicated the presence of an anion binding site. Apparent affinity constants for chloride and thiocyanate ions were calculated to 100 and 50 mM, respectively. In contrast to the effect on the peptidase activity, no chloride-stimulation could be detected of the epoxide hydrolase activity of this enzyme, i.e., the conversion of leukotriene A4 into leukotriene B4. In conclusion, the results indicate that under physiological conditions, chloride ions may selectively stimulate the peptidase activity of LTA4 hydrolase. Also, the differences in chloride concentrations between cellular compartments suggest that a possible proteolytic function of the enzyme may be limited to the extracellular space.     

Types of enzymatic overdosing in trisomy 21: erythrocytic superoxide dismutase-AJ and phosphoglucomutase.

A comparative study was carried out on the hemolysates of 6 trisomic 21 and 6 normal subjects, by electrophoresis in starch gel, determining by a combined staining method both SOD-A (former IPO-dimer) and PGM activity. The enzymes were found statistically to be in a hyperactive status, the ratio of trisomic to normal values being approximately equalt to 1.4. SOD-A supraactivation is the effect of a genic dose, as demonstrated in earlier works (Sichitiu, 1973; Sichitiu et al., 1974; Sinet et al., 1974), whereas PGM hyperactivity appears to be modified secondarily, the same as the activity of other cellular enzymes in Down's disease.     

Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.     

Recombinant Lactococcus starters as a potential source of additional peptidolytic activity in cheese ripening

AIMS: The aim of this study was to modulate the lactococcal proteolytic system for enhancement of the cheese ripening process. METHODS AND RESULTS: The genes encoding PepN, PepC, PepX and PepI peptidases of a highly proteolytic Lactobacillus helveticus strain were transferred into Lactococcus lactis in a food-grade cloning system. A comparison of the relative peptidase activities from the transformants with those from the untransformed host, determined in the conditions of maturing cheese, showed that an increase in peptidase activity could be achieved by introducing a selected peptidase gene from Lact. helveticus into L. lactis. CONCLUSIONS: Recombinant L. lactis starter strains, carrying a peptidase gene from Lact. helveticus, may have an important contribution to the proteolysis of maturing cheese by producing an additional peptidolytic enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be of importance in shortening the ripening period and production of special cheeses (e.g. reduced-fat cheeses) with improved characteristics.     

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.