Unexpected destruction of chitosomal chitin synthetase by an endogenous protease during sucrose density gradient purification

1991 ◽  
Vol 99 (3) ◽  
pp. 565-570
Author(s):  
T. Kamada ◽  
C.E. Bracker ◽  
E. Lippman ◽  
S. Bartnicki-Garcia
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Synthetase Activity ◽  
Phenylmethylsulfonyl Fluoride ◽  
Mucor Rouxii ◽  
Ph Optimum ◽  
Chitin Synthetase ◽  
Endogenous Protease ◽  
Density Gradient Purification

Because of their intrinsic low buoyant density, chitosomes can be separated from crude cell homogenates (1000 g or 35,000 g supernatants) of Mucor rouxii by isopycnic sedimentation in sucrose density gradients. To accelerate and simplify the isolation of chitosomes, we centrifuged the cell-free extracts at ultrahigh speed (in a fixed-angle rotor at forces up to 311,000 g Rav) and found that the duration of centrifugation was critical. Prolonged centrifugation at ultrahigh speed caused severe distortion of the chitin synthetase profile in the gradient as the peak of chitosomal chitin synthetase nearly disappeared. We traced the problem to a soluble protease(s) that moved into the chitosome band during protracted centrifugation and destroyed the chitin synthetase activity. The interfering protease was a soluble protein with a sedimentation coefficient of 4.6 S and a pH optimum of 7–7.5, and it was sensitive to PMSF (phenylmethylsulfonyl fluoride), indicating that it was a serine protease. Unlike other proteases, it destroyed chitin synthetase but did not activate the chitin synthetase zymogen. The interfering protease could be eliminated either by adding PMSF to the cells immediately after breakage or by removing the upper part of the sucrose gradient midway through the centrifugation of the cell-free extract and then completing the sedimentation with the ‘decapitated’ gradient.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

scholarly journals The Isolation and Oharacterization of An Rna Bacteriophage

1964 ◽  
Vol 17 (3) ◽  
pp. 719 ◽  
Author(s):  
CI Davern
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Culture Medium ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Caesium Chloride ◽  
Specific Phage ◽  
K 12 ◽  
Male Specific ◽  
Rna Phage

An enrichment procedure for the isolation of RNA bacteriophage is described. The method involves the inoculation of sewage samples into cultures of Escherichia coli K-12 Hfr under conditions where DNA synthesis is restricted by the addition of 5-fiuorodeoxyuridine to the culture medium. Six phage isolates were made and all of them were shown to be male-specific. One of the male-specific phage was further characterized as an RNA phage, having very similar properties to RNA phage already isolated in other parts oftha world. This RNA phage has a buoyant density of 1�42 g/cm3 in caesium chloride. and has a sedimentation coefficient of 79'5 Sin O'Ol:M Tria-HOI buffer, pH 7� 4, at 20�0.

Disappearance of the covalently closed circular cryptic plasmid PO-2 in Salmonella pullorum MS35 containing F77

1974 ◽  
Vol 20 (4) ◽  
pp. 551-557
Author(s):  
Paul W. Stiffler ◽  
D. E. Schoenhard
Keyword(s):  
Salmonella Typhimurium ◽  
Sucrose Gradient ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Apparent Effect ◽  
Donor Property ◽  
Circular Dna ◽  
Salmonella Pullorum ◽  
Cryptic Plasmids ◽  
Equilibrium Centrifugation

The physical basis of the donor property of Salmonella pullorum donor strains MS8300, MS830, and MS831 carrying the F77 factor from Salmonella typhimurium was investigated by dye-buoyant density equilibrium centrifugation and zonal centrifugation in neutral sucrose gradients. Centrifugation of the MS8300 and MS831 closed circular DNA material in a 20 to 31% neutral sucrose gradient resulted in a profile having one sharp peak of radioactivity with a sedimentation coefficient of 17 S and a broad peak extending from 65 to 70 S. The 17- and 65-S species were isolated from the isogenic F− strain MS83. These appeared identical with those isolated previously in S. pullorum MS53 as the cryptic plasmids PO-1 and PO-2 respectively. Cosedimentation of differentially labeled F77 DNA and the lysate containing the 65-S and 70-S species suggested that the 70-S species is the autonomous F77 factor in strains MS8300 and MS831. Lysates of MS830 similarly treated produced a profile containing the 17-S molecule and possibly some 70-S molecules but no 65-S molecules. It was concluded that the F77 factor was integrated in most cells and that the covalently closed circular state of PO-2 plasmid was lost. The mutation in the cysE gene of the F77 factor carried by MS831 had no apparent effect on the covalently closed circular nature of PO-2 plasmid, although F77 no longer seemed to mobilize the chromosome from the cysE locus.

Is LM7 a new human retrovirus or a mycoplasma virion?

1995 ◽  
Vol 1 (2) ◽  
pp. 88-94 ◽  
Author(s):  
P Froussard
Keyword(s):  
Cell Culture ◽  
Reverse Transcription ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Enveloped Virus ◽  
Polymerase Chain ◽  
Serological Data ◽  
Culture Supernatants ◽  
Viral Genomic Rna ◽  
Human Retrovirus

A putative retrovirus called LM7 was recently isolated from a patient with MS. This retrovirus was detected in LM7 and LM711 cultured human leptomeningeal cells. In the present work, nucleic adds from LM711 cell culture supernatants were purified and subjected to avian myeloblastosis viral (AMV) reverse transcriptase and to random polymerase chain reaction (rPCR) in order to characterize the genomic material of LM7 virions. Analysis of reverse transcription products allowed the detection of an approximately 14 kb ribonucleic acid in all LM711 cell culture supernatants. However, sequencing of rPCR-amplified molecules as well as RNA blotting data showed essentially that all tested cells producing LM7 particles were infected with mycoplasma. Moreover, purification of LM7 particles onto a linear sucrose density gradient established that the 14 kb nucleic acid was always associated with the 1.19–1.21 g ml-1 sucrose fractions, which are known to correspond to the buoyant density of mycoplasma. In addition, no viral genomic RNA was detected in the 1.17 g ml-1 sucrose fraction containing the low reverse transcription activity. These results strongly suggest that microscopic images and serological data could be related to mycoplasma and/or to a virion associated with the bacteria. The LM7 particle might be a new and additional enveloped virus able to infect Mycoplasma hyorhinis hosts. Thus, for instance, it would be presumptuous to assert, with our current understanding, that the LM7 virion is one of the causal agents of MS in humans.

Triton WR-1339 injected into rats enters renal renin granules

1988 ◽  
Vol 255 (3) ◽  
pp. E353-E356
Author(s):  
B. J. Morris
Keyword(s):  
Plasma Proteins ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Extracellular Fluid ◽  
Sucrose Density Gradient ◽  
Equilibrium Density ◽  
Juxtaglomerular Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton Wr 1339 ◽  
Extracellular Substances

To test directly the possibility that substances in extracellular fluid can gain access to renin storage granules in renal juxtaglomerular cells, rats were injected with Triton WR-1339, which binds to plasma proteins. A heavy granule fraction was prepared, and isopycnic sucrose density gradient centrifugation was performed. The renin granule peak was found to be altered from a mean equilibrium density of 1.202 g/ml in control rats to 1.196 g/ml for rats injected with Triton WR-1339 (P less than 0.005). The distribution of angiotensinogen, which is bound in kidney granules having a different buoyant density, was also examined and found to be unaltered. After injection, Triton WR-1339 binds to circulating plasma proteins. The results for renin support the possibility of pinocytotic uptake of protein-Triton WR-1339 complexes by the juxtaglomerular cells with subsequent fusion of the endocytotic lysosomal vacuoles with renin granules accounting for the translocation of ingested substances into the granule matrix. If so, the potential would therefore exist for interaction(s) of ingested extracellular substances with renin or other components in the granules. The present study has therefore demonstrated directly that endogenous extracellular substances may enter renin granules.

scholarly journals Neuroprotection by Neurotropin through Crosstalk of Neurotrophic and Innate Immune Receptors in PC12 Cells

2020 ◽  
Vol 21 (18) ◽  
pp. 6456
Author(s):  
Yu Fukuda ◽  
Kazuki Nakajima ◽  
Tatsuro Mutoh
Keyword(s):  
Pc12 Cells ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Innate Immune ◽  
Membrane Microdomains ◽  
Immune Receptors ◽  
Neurite Retraction ◽  
Ischemic Attack ◽  
Molecular Patterns

Infected or damaged tissues release multiple “alert” molecules such as alarmins and damage-associated molecular patterns (DAMPs) that are recognized by innate immune receptors, and induce tissue inflammation, regeneration, and repair. Recently, an extract from inflamed rabbit skin inoculated with vaccinia virus (Neurotropin®, NTP) was found to induce infarct tolerance in mice receiving permanent ischemic attack to the middle cerebral artery. Likewise, we report herein that NTP prevented the neurite retraction in PC12 cells by nerve growth factor (NGF) deprivation. This effect was accompanied by interaction of Fyn with high-affinity NGF receptor TrkA. Sucrose density gradient subcellular fractionation of NTP-treated cells showed heretofore unidentified membrane fractions with a high-buoyant density containing Trk, B subunit of cholera toxin-bound ganglioside, flotillin-1 and Fyn. Additionally, these new membrane fractions also contained Toll-like receptor 4 (TLR4). Inhibition of TLR4 function by TAK-242 prevented the formation of these unidentified membrane fractions and suppressed neuroprotection by NTP. These observations indicate that NTP controls TrkA-mediated signaling through the formation of clusters of new membrane microdomains, thus providing a platform for crosstalk between neurotrophic and innate immune receptors. Neuroprotective mechanisms through the interaction with innate immune systems may provide novel mechanism for neuroprotection.

A NON-NUCLEOTIDE POLYMER FOUND IN THE DNA OF THE SAND DOLLAR, ECHINARACHNIUS PARMA: II. PRELIMINARY CHARACTERIZATION

1967 ◽  
Vol 45 (2) ◽  
pp. 281-287 ◽  
Author(s):  
Herbert S. Rosenkranz
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Negative Electrode ◽  
Cesium Chloride ◽  
Amino Sugar ◽  
Electrophoretic Analysis ◽  
Positive Test ◽  
Preliminary Characterization ◽  
Acid Hydrolysate

A preliminary characterization of the non-nucleotidic component present in the DNA of Echinarachnius parma was undertaken. This material has an extremely high sedimentation coefficient (907 S). It contains no deoxyribose and presumably no ribose. After acid hydrolysis it was strongly ninhydrin-positive and also gave positive tests for reducing sugars as well as a slightly positive test for amino sugars. Upon electrophoretic analysis of an acid hydrolysate, three ninhydrinpositive spots were detected. One of these migrated to the negative electrode with a mobility identical with that of galactosamine, the other migrated to the positive electrode, and the third was neutral at pH 6.3. The spot with a mobility identical with that of galactosamine also gave a positive test for amino sugar. The material was not attacked by α-amylase. However, digestion with a crude trypsin preparation resulted in loss of the banding property in gradients of cesium chloride. Exposure to purified trypsin did not completely digest it, but caused an increase in buoyant density.

Synthesis of Cell Wall Microfibrils in vitro by a "Soluble" Chitin Synthetase from Mucor rouxii

Science ◽  
1974 ◽  
Vol 186 (4161) ◽  
pp. 357-359 ◽  
Author(s):  
J. Ruiz-Herrera ◽  
S. Bartnicki-Garcia
Keyword(s):  
Cell Wall ◽  
Mucor Rouxii ◽  
Chitin Synthetase

DEMONSTRATION OF A CYTOPLASMIC RECEPTOR PROTEIN FOR OESTROGEN IN THE CANINE PROSTATE GLAND

1978 ◽  
Vol 78 (1) ◽  
pp. 131-139 ◽  
Author(s):  
N. CHAISIRI ◽  
Y. VALOTAIRE ◽  
BRONWEN A. J. EVANS ◽  
C. G. PIERREPOINT
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Prostate Gland ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Receptor Protein ◽  
Receptor Complex ◽  
Binding Properties ◽  
Sucrose Density Gradient Centrifugation ◽  
A Value

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid–receptor complex was found to have a sedimentation coefficient of 4–5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3·8 ± 1·53 (s.d.) × 10−10 mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

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