Activity and location of two enzyme fractions during the culture cycle of Schizosaccharomyces pombe

1970 ◽  
Vol 16 (3) ◽  
pp. 187-191 ◽  
Author(s):  
Gail Dolan Rock ◽  
B. F. Johnson
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Walls ◽  
Membrane Fraction ◽  
Soluble Fraction ◽  
Total Activity ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
E Coli ◽  
Culture Cycle ◽  
Ribonuclease I

When cell walls of Schizosaccharomyces pombe were removed, the protoplast contained most of the ribonuclease but only about 50% of the aminopeptidase activity. In cell homogenates approximately 75% of the total peptidase activity was in the soluble fraction; the membrane fraction retained an average of 25% while the ribosomes had less than 1% of the total activity. The RNase activity was highest in stationary phase, aminopeptidase at mid log phase. Properties of the soluble aminopeptidase were similar in many respects to those of the enzyme from Escherichia coli. In contrast, 90% of the ribonuclease activity was attached to the membrane fraction and up to 10% was found on the ribosomes. The ribosome-bound ribonuclease required incubation in 4 M urea for activation; however, the purified ribonuclease had properties similar to the ribonuclease I of E. coli.

THE LOCALIZATION AND PROPERTIES OF AN AMINOPEPTIDASE IN ESCHERICHIA COLI B

1963 ◽  
Vol 41 (1) ◽  
pp. 9-18 ◽  
Author(s):  
A. T. Matheson
Keyword(s):  
Membrane Fraction ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Progressive Increase ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Phase Growth ◽  
Latent Form ◽  
E Coli ◽  
Escherichia Coli B

Two types of intracellular aminopeptidase activity are present in E. coli B. One type, present in the 'soluble' fraction, is completely inactivated by chymotrypsin or trypsin; the other, in the particulate fractions ('ribosome' and 'membrane'), is resistant to these enzymes. The 'ribosomal' peptidase activity is present partially in a latent form which becomes activated on disruption of the ribosome structure. During the transition from log phase to post-log phase growth there is a progressive increase in the specific activity of the peptidase in the 'soluble' and 'membrane' fraction and a corresponding decrease in the 'ribosome' fraction.

THE LOCALIZATION AND PROPERTIES OF AN AMINOPEPTIDASE IN ESCHERICHIA COLI B

1963 ◽  
Vol 41 (1) ◽  
pp. 9-18 ◽  
Author(s):  
A. T. Matheson
Keyword(s):  
Membrane Fraction ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Progressive Increase ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
Phase Growth ◽  
Latent Form ◽  
E Coli ◽  
Escherichia Coli B

Two types of intracellular aminopeptidase activity are present in E. coli B. One type, present in the 'soluble' fraction, is completely inactivated by chymotrypsin or trypsin; the other, in the particulate fractions ('ribosome' and 'membrane'), is resistant to these enzymes. The 'ribosomal' peptidase activity is present partially in a latent form which becomes activated on disruption of the ribosome structure. During the transition from log phase to post-log phase growth there is a progressive increase in the specific activity of the peptidase in the 'soluble' and 'membrane' fraction and a corresponding decrease in the 'ribosome' fraction.

Localization of the Basic Aminopeptidases in Escherichia coli

1971 ◽  
Vol 49 (12) ◽  
pp. 1340-1346 ◽  
Author(s):  
A. T. Matheson ◽  
L. P. Visentin ◽  
A. Boutet ◽  
C. F. Rollin
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Ribosomal Subunit ◽  
Growth Conditions ◽  
Aminopeptidase Activity ◽  
E Coli ◽  
Ribosomal Particle ◽  
K 12 ◽  
Ribonuclease I ◽  
Similar Growth

The basic aminopeptidase activity was found in both the 30 S and 50 S ribosomal subunit of Escherichia coli B; the specific activity of the enzyme in the 30 S particle was about six times that found in the 50 S particle. Although about 50% of the enzyme was released from E. coli B ribosomes on dissociation into the subunits, this was not observed with E. coli Q-13, a ribonuclease I-less strain. The partition of the basic aminopeptidase activity between the soluble and ribosomal fractions (isolated in 60 mM KCl) of E. coli was determined by the strain of E. coli used, as well as the stage and temperature of growth. Under similar growth conditions, a significantly greater amount of enzyme was found in the ribosomal fraction of E. coli B than in the corresponding fraction of E. coli K-12. When E. coli B cells were disrupted in 300 mM KCl, over 90% of the enzyme was found in the soluble fraction. The remaining 10% on the ribosomal particle had enriched activity towards methionyl peptides suggesting that more than one enzyme is present in the basic aminopeptidase fraction.

scholarly journals PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3437-3447 ◽  
Author(s):  
Dilip Chandu ◽  
Dipankar Nandi
Keyword(s):  
Escherichia Coli ◽  
Sodium Salicylate ◽  
Physiological Role ◽  
Negative Regulator ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
E Coli ◽  
Induced Stress ◽  
Hydrolysis Of

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5αΔpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

scholarly journals Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

2003 ◽  
Vol 185 (6) ◽  
pp. 1817-1824 ◽  
Author(s):  
Peter D. Kim ◽  
Trevor Banack ◽  
Daniel M. Lerman ◽  
Jeremiah C. Tracy ◽  
Johanna Eltz Camara ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Soluble Fraction ◽  
Host Protein ◽  
Broad Host Range ◽  
Terminal Portion ◽  
Inner Membrane ◽  
E Coli ◽  
Amino Terminal ◽  
Broad Host Range Plasmid ◽  
Plasmid Rk2

ABSTRACT The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).

Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology

1995 ◽  
Vol 108 (12) ◽  
pp. 3745-3756 ◽  
Author(s):  
K. Takegawa ◽  
D.B. DeWald ◽  
S.D. Emr
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Acid Protein ◽  
Phosphatidylinositol 3

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

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.

Re-Investigation of the Protein Structure of Coenzyme B12-Dependent Diol Dehydrase

1987 ◽  
Vol 42 (4) ◽  
pp. 353-359 ◽  
Author(s):  
Katsuyuki Tanizawa ◽  
Nobuyoshi Nakajima ◽  
Tetsuo Toraya ◽  
Hidehiko Tanaka ◽  
Kenji Soda
Keyword(s):  
Protein Structure ◽  
Membrane Fraction ◽  
Enzyme Purification ◽  
Soluble Fraction ◽  
Proteolytic Cleavage ◽  
Subunit Structure ◽  
Coenzyme B12 ◽  
Enzyme Preparations ◽  
Protein Components ◽  
Limited Digestion

We have purified diol dehydrase, an adenosylcobalamin-dependent enzyme, from Klebsiella pneumoniae by two different procedures to re-investigate its protein structure; one including its extraction with detergent from the membrane fraction, and the other consisting of only chromato­graphic separations of the soluble fraction. The enzyme preparations obtained by these two methods were different in the subunit structure, but both are identical in molecular weight, and in enzymological and immunochemical properties. In addition, the enzyme preparation obtained from the membrane fraction dissociated reversibly into two dissimilar protein components (F and S) in the absence of substrate, as did the preparation from the soluble fraction. Although the subunit multiplicity of component S might be partly due to proteolytic cleavage during the enzyme purification as revealed by limited digestion with trypsin, component F is not a product of proteolytic cleavage of component S, but a primordial and essential constituent of the enzyme.

scholarly journals Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

1994 ◽  
Vol 5 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S Kornbluth ◽  
B Sebastian ◽  
T Hunter ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinases ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Dual Specificity ◽  
Detergent Extraction

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

Fractionation of carbonic anhydrase activity in Gracilaria sp. (Rhodophyta) and Enteromorpha intestinalis (Chlorophyta): changes in the extracellular activity in response to inorganic carbon levels

2000 ◽  
Vol 27 (12) ◽  
pp. 1161 ◽  
Author(s):  
Jesús R. Andría ◽  
Juan J. Vergara ◽  
J. Lucas Pérez-Lloréns
Keyword(s):  
Carbonic Anhydrase ◽  
Inorganic Carbon ◽  
Soluble Fraction ◽  
Total Activity ◽  
Carbonic Anhydrase Activity ◽  
Enteromorpha Intestinalis ◽  
Short Term ◽  
Carbon Availability ◽  
Species Specific ◽  
Extracellular Activity

The presence of different carbonic anhydrase (EC 4.2.1.1) activities has been investigated in the intertidal macroalgae Gracilaria sp. and Enteromorpha intestinalis (L.) Nees by using fractionation techniques. Activities, measured potentiometrically, were recorded for all fractions in both species, including those containing proteins associated with chloroplast membranes. In Gracilaria sp., most of the total activity was present in the soluble fraction, while similar activities were obtained for all fractions in E. intestinalis. By using inhibitors with a different capacity to enter the cell (acetazolamide and 6-ethoxyzolamide, inhibitors of external and total activity, respectively), a surface-accessible location was indicated for a high proportion of the soluble activity obtained in Gracilaria sp. In E. intestinalis, the inhibitor assays showed a substantial dependence of photosynthesis on intracellular activity. The short-term regulation of the extracellular activity in response to inorganic carbon availability was also examined in both macroalgae. Rapid repression (after 2 h) of the activity was recorded when Gracilaria sp. was transferred from limited to replete carbon conditions, while a fairly constant activity was recorded for E. intestinalis. In contrast, an increase of external activity was obtained for both macroalgae after being transferred to carbon-limited conditions, this response being more pronounced in E. intestinalis. Our results suggest the occurrence of a species-specific carbonic anhydrase system.

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