Physicochemical property of bovine brain 73-kDa stress protein

• 1.1. An approximately 70-kDa protein was purified from bovine brain using an ATP-Sepharose column.
• 2.2. The protein sample was found to contain two proteins (major 73 kDa and minor 72 kDa) on two-dimensional gel electrophoresis.
• 3.3. Antibodies raised against the 73- and 72-kDa proteins cross-reacted with stress-induced HSP73 and HSP72 from HeLa cells, respectively.
• 4.4. Heparin-binding peptides were obtained from trypsin digests of HSP73.

     

Neurohypophysial hormones as molecular evolutionary tracers: investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti

• 1.1. Neurohypophysial hormones of two sturgeon species, Acipenser stellatus and Acipenser guldenstadti, have been purified through molecular sieving on Bio-Gel P4 and reverse-phase high pressure liquid chromatography on Nucleosil C18 columns.
• 2.2. Arginine vasotocin has been identified in both species by its retention time in partition chromatography, amino acid composition and, in the case of A. stellatus, by amino acid sequencing.
• 3.3. A second peptide has been purified and could be α-deamidated vasotocin.
• 4.4. Another peptide with oxytocic activity, distinct from the known oxytocin-like peptides, seems to be present in very small amounts.

     

Localization of HSP90 in rat brain

• 1.1. In rats, HSP90 (90-kDa heat shock protein) was abundant in the brain compared with the liver and kidney. Immunohistochemical studies showed the presence of HSP90 in almost all neurons in the brain.
• 2.2. On immunoblotting using an anti-HSP90 antibody, HSP90 was present in a lower amount in the medulla oblongata and spinal cord.
• 3.3. In the pituitary gland, some of superficial cells of the anterior lobe adjacent to the middle lobe was specifically stained with anti-HSP90 antibody.

     

Stress-induced heat-shock protein synthesis in peripheral leukocytes of turkeys,Meleagris gallopavo

• 1.1. Thermal stress, in vitro and in vivo, induced the synthesis of heat-shock proteins, HSP90, HSP70, and HSP23 in turkey leukocytes.
• 2.2. HSP induction was both temperature- and time-dependent.
• 3.3. Salinity-specific stress proteins were expressed with elevated osmolality in culture medium.

     

Identification of a glycoprotein complex containing a glycogen synthase isozyme in Ascaris Suum obliquely-striated muscle

• 1.1. A glycogen/protein complex which contains the major portion of glycogen synthase activity in Ascaris suum muscle has been purified.
• 2.2. The complex contains two proteins which can be dissociated from a glycoprotein component.
• 3.3. The glycoprotein contains glycogen-like domains and is resistant to trypsin digestion.
• 4.4. The glycogen synthase activity in the purified complex catalyzes glycogen synthesis in the absence of exogenous glycogen, but demonstrates an absolute glucose 6-phosphate requirement for activity.
• 5.5. The data support the hypothesis that this isozyme of glycogen synthase is significantly different from the cyclic AMP-regulated enzyme.

     

The major dog pancreas protein recognized by an antiserum to dog prostate kallikrein is the anionic trypsin

• 1.1. On the basis of its immunoreactivity with a polyclonal antiserum to dog prostate kallikrein in Western blot experiments, a 30 kDa protein was purified from the pancreas of the dog using ion-exchange and gel filtration chromatography.
• 2.2. That protein was identified as the anionic trypsin by its NH₂-terminal amino acid sequence.
• 3.3. The immunoreaction occurred despite an overall amino acid homology which was limited to 39% between the prostate kallikrein and anionic trypsin.
• 4.4. Otherwise, the anti-prostatic kallikrein antiserum was rather specific since it did not react with dog cationic trypsin, dog renal kallikrein and human prostate specific antigen.

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Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

A possible cause of heterogeneity of mammalian apurinic/apyrimidinic endonuclease

• 1.1. Mammalian major apurinic/apyrimidinic (AP) endonuclease, APEX nuclease (M_r 35.4 kDa) was purified from HeLa cells. A hybrid protein (M_r 36.4 kDa), which was expressed in BW2001 strain cells of E. coli, comprising human APEX nuclease headed by 10 additional amino acids was also purified.
• 2.2. The purified preparations were frequently associated with 31-, 33- and 35-kDa peptides having AP endonuclease activity.
• 3.3. The 33- and 35-kDa peptides were suggested to be formed from the hybrid protein or APEX nuclease during their purification processes by proteolytic cleavage with subtilisin-like protease. The 31-kDa peptide was thought to be produced by chemical cleavage of the aspartyl-prolyl bond of APEX nuclease.
• 4.4. The results support the notion that some of AP endonuclease heterogeneity based on the molecular weight difference are caused by proteolytic (and chemical) cleavage of a species of AP endonucleases during the extraction and purification.

     

Current developments in stepwise edman degradation of peptides and proteins

• 1.1. Since Edman's (1950, 1956) first publications about 30 years ago, the stepwise degradation of proteins and peptides is universally performed by protein chemists. We review the mechanism of the chemical reactions, and the different special problems encountered, during degradation and different manual methods of degradation.
• 2.2. We take one example of an alternative method using DABITC manually for the degradation of peptides in order to illustrate the evolution of manual degradation techniques (Chang, 1983).
• 3.3. Possibilities and limits of the liquid phase sequenator of Edman and Begg (1967), solid phase sequencer of Laursen (1975) and gas-liquid sequenator of Hewick et al. (1981) and those of Hunkapiller et al. (1983) are considered in detail.
• 4.4. We describe different procedures for identification of PTH-AA or DABTH-AA: thin layer chromatography, gas chromatography, high performance liquid chromatography, etc., in order to illustrate the evolution of the procedures of identification.
• 5.5. We conclude by taking two manual examples and two automatic procedures of degradation to underline the progress over the last decade.

     

Bayesian Confidence Intervals for Multiplexed Proteomics Integrate Ion-statistics with Peptide Quantification Concordance,

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Highlights

• •Bayesian Beta-Binomial model integrates ion statistics with peptide ratio agreement.
• •Model appropriately interprets information from low signal peptides.
• •Confidence can be assigned even without replicates.
• •Model adds sensitivity to detection of small changes.

     

Human spleen cathepsin D: Its characterization and localization in human spleen

• 1.1. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography.
• 2.2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins.
• 3.3. Inhibition with thiol compounds was found to be a specific phenomenon of the cathepsin D from the human spleen.
• 4.4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel.
• 5.5. In an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen.
• 6.6. The role of this enzyme in human spleen cell was discussed.

     

Analysis of Endogenous Peptides Released from Osteoarthritic Cartilage Unravels Novel Pathogenic Markers,

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Highlights

• •Shotgun identification of neopeptides released from osteoarthritic cartilage.
• •Specific endogenous peptides from the cartilage ECM are measured by MRM.
• •Identification of neopeptides differentially generated from diseased tissue.
• •The peptide DSNKIETIPN shows the best metrics as biomarker of OA cartilage.

     

HLA-DO Modulates the Diversity of the MHC-II Self-peptidome

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Highlights

• •MHC-II-bound peptide repertoires from DO-sufficient and DO-deficient cells.
• •Fewer unique peptides and core epitopes were presented in the absence of DO.
• •Immunopeptidome differences appeared to result from reduced DM editing.
• •DO-dependent self-epitopes elicited CD4 T cell responses in mice.

     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Isolation and partial characterization of a second myotropic peptide from the hindgut of the cockroach,Leucophaea maderae

• 1.1. Proctolin and a second myotropic peptide were extracted from the hindgut of the cockroach Leucophaea maderae with methanol-water-acetic acid (90:9:1). The two peptides were easily separated by HPLC on a μ-Bondapak-phenyl column.
• 2.2. Like proctolin, the second peptide was heat stable and was inactivated by the exopeptidases aminopeptidase M and carboxypeptidase Y.
• 3.3. The response of the isolated hindgut to the new peptide was distinguishable from the response to proctolin by the following features: (a) a longer interval following application (1–4 min) to reach a maximum contraction, and (b) a much larger amplitude for single phasic contractions. Like proctolin, the new peptide could cause a protracted stimulation of the hindgut for more than 2 hr.

     

Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics

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Highlights

• •The developed Ac-LysargiNase showed higher stability and activity than before.
• •The merged spectra of the mirror peptides achieved nearly complete ion coverage.
• •pNovoM obviously increased the efficiency and accuracy of peptide sequencing.
• •The mirror enzymatic strategy achieved precision de novo sequencing on proteome scales.

     

Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples

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Highlights

• •Single-pot workflow for manual or automated enrichment of N-terminal peptides.
• •Sensitive enrichment of protein N termini from 10,000 cells or 2 μg crude proteome.
• •Data independent acquisition improves precision of peptide level quantification.
• •First degradomic analyses of sorted immune cells, single seedlings, and mitochondria from patient cells.

     

Cotranslational fatty acylation of mucus glycoprotein. Addition of palmitic acid to peptidyl-tRNA occurs prior to peptide chain completion and its release

• 1.1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [³H]palmitic acid and [³⁵S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells.
• 2.2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes.
• 3.3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [³H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da.
• 4.4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30–43 amino acids and those of 18–25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10–15 amino acids.
• 5.5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occuring in an immediate vicinity of the signal peptide fragment.