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21.
Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned. 相似文献
22.
The possible inhibition of adenylyl cyclase activity by atrial peptides selective for the ANF-C receptor was investigated in bovine pulmonary artery endothelial cells. In these cells isoprenaline, guanine nucleotide and forskolin dose-dependently increased activity over basal levels. In the presence of rANF(99-126), these dose-dependent increases were not reduced, nor were they affected by the ANF-C receptor selective analogue C-ANF(102-121). Furthermore, the selective analogues rANF(103-123) and des[Cys105,Cys121]rANF104-126 had no effect on basal or stimulated adenylyl cyclase activity. It can be concluded that ANF-C receptors are not linked to inhibition of adenylyl cyclase in these cells. 相似文献
23.
Rat liver glucokinase (EC 2.7.1.2) is a monomeric enzyme with positive cooperativity for glucose phosphorylation for which several kinetic mechanisms have been proposed. We have observed a slow kinetic transition when the enzyme is assayed in the presence of 30% glycerol. When the enzyme had been preincubated or stored in 50 mM glucose, the initially rapid activity decayed, via a first-order process, to a new steady-state velocity. The glucose-induced process is reversible since if the enzyme is preincubated without glucose, an initially low activity accelerates over minutes to the same steady-state velocity. This final velocity is independent of the preincubation conditions and is determined solely by the glucose and ATP concentrations in the assay. Possible artifacts which might cause nonlinear progress curves have been ruled out. The transition has a half-time of 2-10 min depending on glucose and ATP concentrations and temperature. In the steady-state kinetics, positive cooperativity occurs with glucose with a Hill coefficient (nH) = 1.3 at high ATP concentrations, approaching unity as the ATP concentration decreases. This pattern is similar to that seen in the linear velocities in the absence of glycerol. Similarly, negative cooperativity with MgATP is seen in the steady-state velocities at low glucose concentrations with the Hill coefficient approaching 1 as the glucose concentrations approach saturation. The initial velocity for enzyme preincubated in high glucose concentration was either Michaelis-Menten as a function of glucose at high MgATP concentration or heterogeneous (nH less than 1, negatively cooperative) at low MgATP concentration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
24.
Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides 总被引:2,自引:0,他引:2
The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells. 相似文献
25.
Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay. 相似文献
26.
Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland
Summary Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and were located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 g/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis. 相似文献
27.
Undecaprenyl pyrophosphate synthetase was partially purified from Lactobacillus plantarum by DEAE-cellulose, hydroxyapatite, and Sephadex G-100 chromatography in Triton X-100. The enzyme has a molecular weight between 53,000 and 60,000. The enzyme demonstrated a fivefold preference for farnesyl pyrophosphate rather than geranyl pyrophosphate as the allylic cosubstrate, whereas dimethylallyl pyrophosphate was not effective as a substrate. Polyprenyl pyrophosphates obtained using either farnesyl or geranyl pyrophosphate as cosubstrate were chromatographically identical. Hydrolysis of these polyprenyl pyrophosphates with either a yeast or liver phosphatase preparation yielded undecaprenol as the major product. Incorporation of radioactive label from mixtures of Δ3-[1-14C]isopentenyl pyrophosphate and Δ3-2R-[2-3H]isopentenyl pyrophosphate into enzymic product indicated that each isoprene unit added to the allylic pyrophosphate substrate has a cis configuration about the newly formed double bond. The removal of detergent from enzyme solutions resulted in a parallel loss in enzyme activity when analyzed with either farnesyl or geranyl pyrophosphate as cosubstrates. Enzymic activity was restored on addition of Triton X-100 or deoxycholate. The enzyme exhibited a pH-activity profile with optima at pH 7.5 and 10.2. It also demonstrated a divalent cation requirement, with Mg2+, Mn2+, Zn2+, and Co2+ exhibiting comparable activities. 相似文献
28.
Ganglioside in the membrane of erythrocytes and the fat globules of cow's milk were not degraded by neuraminidase treatment at pH 5.2 or 7.4. Removal of portions of the membrane protein by treatment with trypsin or by extraction with a solution of EDTA and 2-mercaptoethanol rendered these membrane-associated gangliosides accessible to neuraminidase attack. After trypsin treatment under conditions where the membranes are impermeant to this enzyme, gangliosides were exposed to neuraminidase. The results suggest that the carbohydrate groups of the gangliosides of bovine erythrocytes and milk fat globules are located primarily on the environmental face of the membrane and are shielded from neuraminidase attack by membrane proteins. 相似文献
29.
Roy W. Keenan Michael Kruczek Lynn Fusinato 《Archives of biochemistry and biophysics》1975,167(2):697-705
Cell-free preparations from Tetrahymena pyriformis catalyze the incorporation of glucose from UDP-glucose into a glucolipid having properties which are identical to those of other dolichyl phosphoryl sugar derivatives. Kinetic and other experiments have provided evidence that this glucolipid serves as glucose donor for two other types of glucosylated substances, one of which has been tentatively identified as an oligosaccharide lipid and the other a glycoprotein or glycoproteins. In addition, the partially purified glucolipid served as a glucosyl donor to these cell components, suggesting that in this protozoan, at least part of the glycoprotein is synthesized by reactions involving lipid-linked sugars in a manner analogous to that which has been observed in glycoprotein synthesis in mammalian cells. 相似文献
30.
Specific activities of diacylglycerol acyltransferase, glycerol 3-phosphate acyltransferase and pyruvate dehydrogenase were studied in virgin, pregnant, lactating and involuting rat mammary glands. An inverse relationship was evident between cAMP binding to protein kinase(s) and the activities of the above enzymes in lactating rat mammary glands. Results suggested that free Ca2+ concentration may also contribute to control of the activity of pyruvate dehydrogenase in these glands. However, no consistent change was observed between the activities of these enzymes and cAMP binding in young, pregnant and involuting rat mammary glands. Calmodulin levels paralleled bound Ca2+ except in lactating rats. Almost all parameters studied peaked on day 8 of lactation. 相似文献