Organization of enzymes in the synthesis of peptides

1954 ◽  
Vol 142 (907) ◽  
pp. 170-174 ◽  
Keyword(s):  
Amino Acid ◽  
Leucine Aminopeptidase ◽  
Mitotic Rate ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Exchange Reactions ◽  
Different Types ◽  
A Cell ◽  
Coupled Reactions ◽  
Hydrolysis Of

We have undertaken the investigation of the nature of the intracellular peptidases and transpeptidases in the belief that the information will be pertinent to an understanding of the genetic behaviour and growth of an organism or, simply, the synthesis of protein. It cannot be argued that the peptidases and transpeptidases are responsible for all steps in protein synthesis; the participation of peptidases in the synthesis of peptides and proteins would require that energy be derived from coupled reactions. Nevertheless, it would seem obvious that the peptidases are concerned with the synthesis of protein; peptidase activity is greatest in rapidly growing tissue and may be correlated with the mitotic rate of various types of cells. In addition, it is easily demonstrated that the peptidases can, in the presence of a suitable linked source of energy, catalyze the formation of dipeptides. Energy mechanisms are required, but these energy mechanisms are probably coupled with exchange reactions of many different types and specificities; these specificities may well be furnished by the peptidases. Our studies have indicated that the peptidase activities of a cell are a property of the polynucleotides of the cell (Binkley 1952). In our studies of the hydrolysis of glutathione (Olson & Binkley 1950) it became necessary to study the hydrolysis of the cysteinylglycine. Our most highly purified preparations of the enzyme have been found to be non-specific in nature and to hydrolyze all dipeptides not having an amino-acid of the D-configuration as the initial amino-acid. Leucylglycine, a substrate for the so-called leucine aminopeptidase activity of cells, is easily prepared and is an excellent substrate for studies of the non-specific dipeptidase; this substrate has been used in all our more recent work.

Quantification of small-scale heterogeneity in aquatic aminopeptidase activity

2020 ◽  
Vol 84 ◽  
pp. 127-140
Author(s):  
BM Gaas ◽  
JW Ammerman
Keyword(s):  
Chlorophyll Fluorescence ◽  
Leucine Aminopeptidase ◽  
Hudson River ◽  
Aminopeptidase Activity ◽  
Small Scale ◽  
Analysis Instrument ◽  
Sequential Samples ◽  
Degree Of Confidence ◽  
The Mean ◽  
Hydrolysis Of

Leucine aminopeptidase (LAP) is one of the enzymes involved in the hydrolysis of peptides, and is sometimes used to indicate potential nitrogen limitation in microbes. Small-scale variability has the potential to confound interpretation of underlying patterns in LAP activity in time or space. An automated flow-injection analysis instrument was used to address the small-scale variability of LAP activity within contiguous regions of the Hudson River plume (New Jersey, USA). LAP activity had a coefficient of variation (CV) of ca. 0.5 with occasional values above 1.0. The mean CVs for other biological parameters—chlorophyll fluorescence and nitrate concentration—were similar, and were much lower for salinity. LAP activity changed by an average of 35 nmol l-1 h-1 at different salinities, and variations in LAP activity were higher crossing region boundaries than within a region. Differences in LAP activity were ±100 nmol l-1 h-1 between sequential samples spaced <10 m apart. Variogram analysis indicated an inherent spatial variability of 52 nmol l-1 h-1 throughout the study area. Large changes in LAP activity were often associated with small changes in salinity and chlorophyll fluorescence, and were sensitive to the sampling frequency. This study concludes that LAP measurements in a sample could realistically be expected to range from zero to twice the average, and changes between areas or times should be at least 2-fold to have some degree of confidence that apparent patterns (or lack thereof) in activity are real.

scholarly journals Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 462-469 ◽  
Author(s):  
RA Ashmun ◽  
AT Look
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Aminopeptidase N ◽  
Surface Glycoprotein ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Cell Surface Glycoprotein ◽  
Metalloprotease Inhibitors

Abstract We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p- nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2′- dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13- positive cells and was blocked by the metalloprotease inhibitor 1,10- phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

1991 ◽  
Vol 58 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Teresa Requena ◽  
Carmen Peláez ◽  
Michel J. Desmazeaud
Keyword(s):  
Leucine Aminopeptidase ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Titratable Acidity ◽  
Aminopeptidase Activity ◽  
Whole Cells ◽  
Alanine Aminopeptidase ◽  
Triton X ◽  
Hydrolysis Of

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

scholarly journals Input of Protein to Lake Water Microcosms Affects Expression of Proteolytic Enzymes and the Dynamics ofPseudomonas spp

2001 ◽  
Vol 67 (11) ◽  
pp. 4955-4962 ◽  
Author(s):  
Jakob Worm ◽  
Ole Nybroe
Keyword(s):  
Population Structure ◽  
Lake Water ◽  
Leucine Aminopeptidase ◽  
Proteolytic Enzymes ◽  
Extracellular Enzyme ◽  
Bacterial Consortium ◽  
Proteinase Activity ◽  
Aminopeptidase Activity ◽  
Growth Media ◽  
Hydrolysis Of

ABSTRACT The objective of this study was to determine how an input of protein to lake water affects expression of a proteolytic potential and influences the abundance and composition of a specific group of bacteria. Pseudomonas spp. were chosen as a target group that can be recovered on selective growth media and contain both proteolytic and nonproteolytic strains. Amendment with 2 mg of casein per liter increased total proteinase activity (hydrolysis of [3H]casein) by 74%, leucine-aminopeptidase activity (hydrolysis of leucine-methyl-coumarinylamide) by 133%, bacterial abundance by 44%, and phytoplankton biomass (chlorophylla) by 39%. The casein amendment also increased the abundance of culturable Pseudomonas spp. by fivefold relative to control microcosms but did not select for proteolytic isolates. Soluble proteins immunochemically related to thePseudomonas fluorescens alkaline proteinase, AprX, were detected in amended microcosms but not in the controls. The expression of this class of proteinase was confirmed exclusively for proteolyticPseudomonas isolates from the microcosms. The population structure of Pseudomonas isolates was determined from genomic fingerprints generated by universally primed PCR, and the analysis indicated that casein amendment led to only minor shifts in population structure. The appearance of AprX-like proteinases in the lake water might thus reflect a general induction of enzyme expression rather than pronounced shifts in the Pseudomonaspopulation structure. The limited effect of casein amendment onPseudomonas population structure might be due to the availability of casein hydrolysates to bacteria independent of their proteinase expression. In the lake water, 44% of the total proteinase activity was recovered in 0.22-μm-pore-size filtrates and thus without a direct association with the bacteria providing the extracellular enzyme activity. Since all Pseudomonasisolates expressed leucine-aminopeptidase in pure culture, proteolytic as well as nonproteolytic pseudomonads were likely members of the bacterial consortium that metabolized protein in the lake water.

scholarly journals Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 462-469 ◽  
Author(s):  
RA Ashmun ◽  
AT Look
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Aminopeptidase N ◽  
Surface Glycoprotein ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Cell Surface Glycoprotein ◽  
Metalloprotease Inhibitors

We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p- nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2′- dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13- positive cells and was blocked by the metalloprotease inhibitor 1,10- phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.

Hormonal and metabolic influences on intracellular peptidase activity

1959 ◽  
Vol 197 (5) ◽  
pp. 1063-1069 ◽  
Author(s):  
Herbert G. Rose ◽  
Mary C. Robertson ◽  
Theodore B. Schwartz
Keyword(s):  
Protein Metabolism ◽  
Leucine Aminopeptidase ◽  
Regulatory Function ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Saline Injection ◽  
Rat Diaphragm ◽  
Peptide Substrate ◽  
Enzyme Adaptation ◽  
Low Levels

A procedure which permits the estimation of intracellular peptidase activity of surviving rat diaphragm has been evaluated following changes in the hormonal and metabolic environment. It was found that leucine aminopeptidase (LAP) activity fluctuated with the seasons, rising during the summer and decreasing to low levels during the winter. LAP activity was significantly increased after acute and chronic fasting, cortisone administration and triiodothyronine administration. Conversely, LAP activity was reduced following adrenalectomy or thyroidectomy. The ‘stress’ of saline injection did not provoke significant changes in aminopeptidase activity and attempts to induce enzyme adaptation by injection of peptide substrate were unsuccessful. These data are consistent with the view that intracellular peptidases perform an important regulatory function in the control of protein metabolism. The possibility has been raised that the changes noted may be the result of varying degrees of intracellular exposure of substrate to enzyme.

scholarly journals Peptidase Activity of Amidinated Thrombin

1977 ◽  
Author(s):  
E.P. Kang
Keyword(s):  
Amino Acid ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Peptidase Activity ◽  
Amino Acid Residues ◽  
Small Peptides ◽  
Human Thrombin ◽  
Small Change ◽  
Hydrolysis Of ◽  
Hydrolysis Activity

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.

scholarly journals The sites of hydrolysis of dipeptides containing leucine and glycine by rat jejunum in vitro

1969 ◽  
Vol 114 (4) ◽  
pp. 855-861 ◽  
Author(s):  
E. B. Fern ◽  
R. C. Hider ◽  
D. R. London
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
The Other ◽  
Peptidase Activity ◽  
Rat Jejunum ◽  
Effective Inhibitor ◽  
Inhibitory Effects ◽  
Molar Basis ◽  
Hydrolysis Of

1. The effect of peptides containing leucine and glycine on accumulation of leucine and glycine by everted jejunal rings was studied. 2. It was shown that, on a molar basis, leucyl-leucine is a more effective inhibitor of uptake of [14C]leucine than is either leucylglycine or glycyl-leucine. These latter dipeptides behave alike. 3. The concentration of the dipeptides and their constituent amino acids in both the incubation medium and the tissue has been followed in these experiments by amino acid analysis. No leucine-containing peptides were observed in the tissue. 4. The inhibitory effects of the mixed dipeptides are altered by pH changes in an analogous way to the alterations in peptidase activity. 5. The experimental results indicate that leucine-containing peptides are hydrolysed before the transport step. 6. Glycylglycine, on the other hand, has only a small effect on the accumulation of glycine, although large amounts of the peptide accumulate unchanged in the tissue. This suggests that glycylglycine is taken up by a different mechanism to that for the leucine dipeptides.

Hydrolysis of Phthalyl Amino Acid Esters of Fluoroscein in the Presence of Leucine Aminopeptidase

1972 ◽  
Vol 140 (1) ◽  
pp. 179-182 ◽  
Author(s):  
J. J. Thomas ◽  
W. C. Eveland ◽  
E. L. Medzon ◽  
W. Christian ◽  
D. E. Wylie ◽  
...  
Keyword(s):  
Amino Acid ◽  
Leucine Aminopeptidase ◽  
Amino Acid Esters ◽  
Hydrolysis Of ◽  
Acid Esters

ACTION OF PROTEOLYTIC ENZYMES ON SOIL ORGANIC MATTER

1970 ◽  
Vol 50 (2) ◽  
pp. 233-241 ◽  
Author(s):  
F. J. SOWDEN
Keyword(s):  
Amino Acids ◽  
Organic Matter ◽  
Amino Acid ◽  
Soil Organic Matter ◽  
Acid Hydrolysis ◽  
Leucine Aminopeptidase ◽  
Proteolytic Enzymes ◽  
Complexing Agent ◽  
Organic Matter Fractions ◽  
Hydrolysis Of

The amino acids set free by proteolytic enzymes were determined with an amino acid analyzer. Soil and enzyme blanks were subtracted. Pronase released 2 to 10% of the aspartic acid + asparagine, threonine, serine, glutamic acid + glutamine, glycine, lysine and histidine in some fractions of soil organic matter along with 15–35% of the alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and arginine. There was no release of proline, ornithine or ammonia. When the pronase hydrolysate was treated with leucine amino-peptidase, 15% of the proline was released, the yield of glycine was raised from 2 to 14% and the amount of the acidic amino acids was also higher. Acid hydrolysis of the pronase hydrolysate also released more amino acid material but the blanks were much higher than with leucine aminopeptidase. The results suggested that more than half of the aspartic and glutamic acids found on acid hydrolysis were present in the soil organic matter fractions as asparagine and glutamine. The action of pronase on the organic matter of the intact soil was slight, even in the presence of a complexing agent. Papain released very little amino acid material from organic matter fractions, but leucine aminopeptidase or HCl hydrolysis of the papain hydrolysate released about 10% of the amino acid of the fraction, indicating that significant amounts of peptides were formed on papain treatment.

Sign in / Sign up

Export Citation Format

Share Document