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1.
Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A 4 to a mixture of leukotrienes C 4 and D 4 in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein ?1 min ?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme. 相似文献
2.
The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1′-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of ‘marker’ enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C 16 alkenyl groups were hydrolysed more rapidly than C 18 alkenyl groups. With 3H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C 14C 18 aldehydes ( Km, 2 μM). 相似文献
3.
Appreciable amounts of free and bound hydroxyproline were found in the tissue fluid obtained by centrifugation from calf costal cartilage. In the tissue fluid the presence of an enzyme capable of splitting synthetic collagenase substrate at pH 7.4 and 37° was also demonstrated. The enzyme has been partially purified by (NH 42SO 4 saturation. Peak activity was obtained in the fraction of 50–60% saturation. The enzyme was not capable of degrading insoluble bovine. Achilles tendon collagen. In our opinion the enzyme cannot be considered a collagenase, and we call its activity “collagenase-like peptidase activity”. 相似文献
4.
The distribution of C 14 was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C 14 in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C 14 in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C 14, glucose-1-C 14, and glucuronolactone-U-C 14, was administered. The highest values were obtained after the administration of glucose-6-C 14; glucuronolactone-U-C 14 gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C 14 assays and autoradiograms indicate that C 14 is also present in other parts of the comb. As seen in autoradiography, the concentration of C 14 was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies, suggest that the high specific activity of carbon in the mucoid layer is due mainly to the presence of C 14-labelled hyaluronic acid. Autoradiograms and PAS staining suggest that a significant amount of C 14 is also incorporated into the glycoproteins associated with the collagen fibers. 相似文献
5.
Collagen metabolism was studied in degenerative articular cartilage of dogs with spontaneous, early onset osteoarthritis. A fraction of collagen which represented about 1.5.% of the total was extracted from cartilage samples with dilute phosphate buffer (pH 7.4) containing 0.2% sodium dodecyl sulfate. Agarose gel filtration in the presence of sodium dodecul sulfate revealed that extracts of degenerative cartilage had about 24% procollagen whereas extracts of normal samples had only 3%. The isolated procollagen fraction was rechromatographed on agarose columns in the presence of mercaptoethanol. This resulted in the identification of a collagen species which migrated between marker β and α collagen chains. The molecular weight of this collagen was estimated to be 150000. Based on incorporation of [ 14C]proline, its ratio of hydroxy[ 14C]proline to total 14C was 0.32. Procollagen was not found after limited pepsin digestion (pH 3,4°C, 16 h) of degenerative cartilage samples.Since the total collagen content (μg hydroxyproline/mg cartilage), hydroxy[ 14C]proline/mg cartilage, specific radioactivity of hydroxy[ 14C]proline (cpm/μg), in the whole cartilage, and the specific radioactivity of hydroxyproline in the extractable collagen fraction were similar for normal and degenerative cartilage we propose that procollagen accumulated in the degenerative cartilage due to a partial defect in conversion of procollagen to collagen. 相似文献
6.
After incubation of [ 4- 14C )progesterone with cell-free homogenates of mouse mammary gland in the presence of NADPH, [ 14C]-labeled 4-pregnene-3α, 20α-diol was identified as a metabolite, besides 20α-hydroxy-4-pregnen-3-one which was the major metabolite. 相似文献
7.
Mutants unable to perform de novo biosynthesis of purines have been isolated from cultures of mutagen-treated Chinese hamster ovary cells using bromodeoxyuridine selection techniques. Accumulation of C 14 -labeled formylglycinamide ribotide by suspension cultures of mutant cells incubated with glycine-C 14 suggested that the defect leading to auxotrophy most probably involves the gene coding for formylglycinamide amidotransferase, (E.C. 6.3, 5.3), the fourth enzyme in the de novo purine biosynthetic pathway. Direct assay of formylglycinamide amidotransferase activity in cell-free extracts prepared from mutant and parental cells has demonstrated the absence of amidotransferase activity in mutant derived extracts. 相似文献
8.
Pancreatic islets were isolated from Wistar rats, albino mice, spiny mice and sand rats ( Psammomys obesus). Evidence is presented that pancreatic islets contain an enzyme system degrading insulin in the presence of glutathione or other sulfhydryl-containing compounds. Apparent Km values for insulin and glutathione (in the presence of EDTA) are 14.0 μM (mol. wt 5700) and 1.28 mM, respectively. Maximum breakdown of 125I-labeled insulin was found at about pH 7.2. After ultracentrifugation of islet homogenates the microsomal fraction contained the greatest relative specific insulin-degrading activity. The specific insulin-degrading actvitity was found to be higher in Wistar rats and albino mice than in spiny mice and sand rats. Starvation of Wistar rats for 72 h caused a decrease inthe enzymatic activity. 相似文献
9.
Intact cells and crude homogenates of high (1% CO 2) and low dissolved inorganic carbon (C i) (30-50 microliters per liter of CO 2) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of 18O from CO 2 to H 2O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low C i cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO 2. This activity is most apparent in the light, where 18O-labeled CO 2 species are being taken up by the cells but the effluxing CO 2 has lost most of its label to water. In the dark, low C i cells catalyze the depletion of the 18O enrichment of CO 2 and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the C i pump and the C i pump is proposed as a potential site for the exchange of 18O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low C i cells. This was inhibited by ethoxyzolamide with an I 50 of 50 to 100 micromolar in both low and high C i cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg 2+ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction. 相似文献
10.
Cathepsin K is responsible for the degradation of type I collagen in
osteoclast-mediated bone resorption. Collagen fragments are known to be
biologically active in a number of cell types. Here, we investigate their
potential to regulate osteoclast activity. Mature murine osteoclasts were
seeded on type I collagen for actin ring assays or dentine discs for
resorption assays. Cells were treated with cathepsins K-, L-, or
MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The
presence of actin rings was determined fluorescently by staining for actin. We
found that the percentage of osteoclasts displaying actin rings and the area
of resorbed dentine decreased significantly on addition of cathepsin
K-digested type I collagen or bone fragments, but not with cathepsin L or
MMP-1 digests. Counterintuitively, actin ring formation was found to decrease
in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin
K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP
inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of
the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted
in an increased actin ring presence in cathepsin K-deficient osteoclasts.
These studies suggest that cathepsin K interaction with type I collagen is
required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial
attachment of osteoclasts and 2) termination of resorption via the creation of
autocrine signals originating from type I collagen degradation.Osteoclasts are monocyte-macrophage lineage-derived, large multinucleated
cells. They are the major bone resorbing cells, essential for bone turnover
and development. Active osteoclasts display characteristic membranes,
including the ruffled border, attachment zone, and the basolateral secretory
membrane. After attachment to bone, the ruffled border secretes enzymes and
protons enabling the solubilization and digestion of the bone matrix.
Osteoclasts express many proteases including cathepsins and matrix
metalloproteases
(MMPs) 2 (for review
see Refs.
1- 3).
However, it is the general consensus that cathepsin K (catK) is the major
bone-degrading enzyme
( 4- 7).Rapid cytoskeletal reorganization is essential for osteoclast function and
formation of the specialized membranes. Bone resorption occurs within the
sealing zone, which is formed by an actin ring structure. This can be
identified as a solid circular belt like formation and consists of an actin
filament core surrounded by actin-binding proteins such as talin,
α-actinin, and vinculin, which link matrix-recognizing integrins to the
cytoskeleton ( 8). The ruffled
border is contained within this structure. The actin ring is initiated by the
formation of podosomes, which represent dot-like actin structures of small
F-actin containing columns surrounded by proteins also found in focal adhesion
such as vinculin and paxillin
( 9). It was previously thought
that the sealing zone was formed by the fusion of podosomes after the
osteoclast becomes activated
( 10,
11), but it has since been
demonstrated that podosomes and the sealing zone are distinct structures
( 12,
13). It should be noted that
bone resorption only occurs when the sealing zone is formed and the actin ring
is present ( 14).Osteoclasts bind and interact with the bone surface through specific
integrin receptors. The most abundant integrin present in osteoclasts is the
αvß3 receptor also known as the vitronectin receptor
( 15,
16). This receptor attaches to
RGD sequence containing components of the bone matrix, e.g.
vitronectin, osteopontin, and type I collagen
( 17- 19).
This interaction enables the formation and regulation of the actin ring and
therefore osteoclast activity
( 20- 22).
It has previously been shown that soluble RGD containing peptides added to
cell supernatant are capable of inhibiting osteoclast binding and bone
resorption ( 18,
22- 24).This study investigates the effect of collagen degradation fragments on
osteoclast activity. Soluble type I collagen and the bone powder of murine
long bones were subjected to digestion reactions by the cysteine proteases,
catK and catL, and the interstitial collagenase, MMP-1. The effect of these
degradation products on osteoclasts was investigated by monitoring actin ring
and resorption pit formation. We further investigated the role of cathepsins
using catK- and catL-deficient mice. Finally, we looked in more detail at the
effect of collagen, as a cell adhesion matrix, on osteoclast activity. 相似文献
11.
1. The C 14O 2 production by Arbacia eggs and embryos from glucose-1-C 14, glucose-2-C 14, and glucose-6-C 14 has been measured without and with dinitrocresol in the incubation medium. In the absence of the dinitrocresol, the C 14O 2 production from glucose-1-C 14 is more rapid than from glucose-2-C 14 and much more rapid than from glucose-6-C 14; this, together with previous findings, indicates that glucose is utilized in Arbacia eggs predominantly via the TPN shunt rather than via the aldolase step of the glycolytic pathway. In the presence of the dinitrocresol, C 14O 2 from glucose-6-C 14 approaches that from glucose-1-C 14, indicating that, in the presence of this reagent, glucose utilization is diverted from the shunt to the glycolytic pathway. 2. Incorporation of C 14 from glucose labelled in the 1-, 2-, or 6- positions into other metabolic products of the eggs and embryos is also inhibited by dinitrocresol, particularly incorporation into the acid-insoluble fraction containing nucleoproteins. 相似文献
12.
To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg efnb1). Col3.6-Tg efnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg efnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg efnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg efnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation. 相似文献
13.
The products of C 14O 2 fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C 14 was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C 14 was found in glycolate products and only a trace of C 14 was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C 14 was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C 14 accumulated in phosphoglycerate. C 14 labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C 14 was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle. Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC14O3 was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C14 accumulated in glycolate. 相似文献
14.
Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo. 相似文献
15.
The plasma membrane fractions from separated cortex and stele of primary roots of corn ( Zea mays L. WF9 × M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP·Mg, and showed complex kinetic data for K + stimulation similar in character to the kinetic data for K +-ATPase and K + influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells. ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K+ stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 μg/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps. 相似文献
16.
The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C 14-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C 14-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells. 相似文献
17.
1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [ 35S]methionine, [ 14C]isoleucine or [ 14C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both 3H and 14C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [ 14C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur. 相似文献
18.
A rapid, sensitive collagenase assay has been developed using 14C-acetylated collagen as a substrate. Acid-soluble calfskin collagen was labeled with [1- 14C]acetic anhydride at pH 8. The acetylated collagen had a specific activity of 6.25 × 10 5 dpm/mg protein. Collagen was not denatured as evidenced by its resistance to nonspecific proteolysis and sensitivity to bacterial collagenase. Polyacrylamide gel electrophoresis of the acetylated protein showed that the radioactivity was present in the three bands corresponding to the α, β, and γ components of collagen. The rate of release of 14C from labeled collagen by Clostridium histolyticum collagenase was proportional to enzyme and substrate concentration. 相似文献
19.
The major C14-labeled substance in sieve tube exudate of M. pyrifera is D -mannitol, comprising 3.6% (w/v). No sugars are detectable. Certain amino acids also possess some C14-labeling and occur in significantly high concentrations in exudate. The exudate contains negligible ether-soluble lipid, but has a large amount of protein and a high concentration of K+ Neither protein nor lipid become labeled significantly in sieve tubes during short-term translocation experiments with C14. In general the chemical composition of the assimilate stream is comparable to that of vascular plants and does not, consequently, necessitate a different mechanism for translocation. 相似文献
20.
The amphiploid Nicotiana suaveolens × N. langsdorffii, which is genetically constituted to produce tumors spontaneously late in development, can be induced to form them in the early seedling stage by treatment of the apical meristem with kinetin and indoleacetic acid. Application of H 3-or C 14-labeled plant-growth regulators resulted in most plants and notably with C 14-labeled indoleacetic acid in a significant increase in the rate of tumor production over growth-regulator treatment alone. Endogenous radiation alone was shown to be tumefacient since radioactive nucleosides, tritiated water, and C 14-sodium carbonate also enhance tumorization. 相似文献
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