Location of glycerol phosphate acyltransferase in the transverse plane of mitochondrial outer membrane of guinea pig lung

Incubation of guinea pig lung mitochondrial suspension in an isotonic low ionic strength buffer containing various proteolytic enzymes caused significant stimulation of the glycerophosphate acyltransferase activity. The maximal stimulation range between 20 and 105%, and the order was as follows: bromelain greater than chymotrypsin greater than pronase greater than trypsin greater than papain greater than nagarse. Under hypotonic conditions, over 85% of GAT was destroyed by all the proteolytic enzymes. Microsomal enzyme activity was consistently inhibited (greater than 95%) by exposure to any of these proteases even under isotonic conditions. These results suggest that GAT is located on the inner aspect of the mitochondrial outer membrane. Also, it is likely that a portion of this enzyme or that of a modulator is present in the outer side of the outer membrane and proteolysis of this component causes stimulation.     

Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.     

Mechanism of neurotensin-induced pressor effect and tachycardia in guinea pigs

Neurotensin (NT) was found to produce a dose-dependent increase of the systolic and diastolic blood pressure, and of the heart rate in anesthetized guinea pigs when injected intravenously (i.v.) as a bolus, or when infused i.v. over a 15 min period. In a small percentage (20%) of animals, bolus injections of NT evoked triphasic variations (e.g. increase followed by a decrease and a further increase) of the blood pressure associated with unpredictable changes of heart rate. The pressor effect of NT was consistently reduced by prior treatment of the animals with pentolinium, a ganglion blocking agent, a mixture of alpha and beta adrenergic receptor blocking drugs, reserpine, a drug known to deplete adrenergic neurons of their neurotransmitters, or guanethidine, a drug known to paralyse adrenergic neurons. NT-induced tachycardia was either unchanged or slightly potentiated following the administration of the latter autonomic blockers. Neither the pressor effect nor the tachycardia evoked by NT was affected by antihistaminics, antiangiotensin or by indomethacin, an inhibitor of prostaglandin synthesis. These results suggest that the pressor effect of NT in anesthetized guinea pigs is likely the result of an interaction (most likely an activation) between the peptide and the sympathetic nervous system. The increase of heart rate induced by NT appears to be due to a direct effect on the heart.     

Loss of target-cell plasma-membrane protein induced by sensitized allogeneic lymphocytes

Sensitized allogeneic aggressor lymphocytes caused a marked loss of plasma-membrane protein in target L cells of a subline sensitive to lymphocyte- or lymphotoxin-induced lysis. By sodium dodecyl sulfate-polyacrylamide electrophoretic analysis, this loss was shown to be general and not restricted to specific membrane fractions. Target L cells of a subline partially resistant to attack by lymphocytes or lymphotoxin showed little or no loss of membrane protein after incubation with sensitized lymphocytes. These observations suggest that target-cell plasma-membrane damage plays an important role in lymphocyte-mediated cytolysis.     

Fluorescence spectra of luteinizing hormone releasing hormone,a decapeptide containing histidyl,tryptophyl and tyrosyl residues: Analysis by derivative spectroscopy

Fluorescence spectra have been obtained for luteinizing hormone releasing hormone, a decapeptide containing His, Trp and Tyr, and analogs lacking one or more of these residues. The second derivatives of these spectra were used to examine the contributions of the three residues to the spectrum of the hormone. Tyr influences the excitation spectrum when fluorescence is monitored at an emission wavelength of 305 nm but makes little or no contribution to the emission spectrum when the compound is excited at 275 nm. His and Trp influence both excitation and emission spectra.     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

Prostaglandin levels in isolated brain microvessels and in normal and norepinephrine-stimulated cat brain homogenates

The levels of PGD₂, PGE₂, PGF_2α and 6-keto-PGF_1α (6KF_1α) produced from endogenous archidonic acid (AA) were quantitated in cat cerebral cortical homogenates and microvessels isolated from cat cerebral cortex using gas chromatography/mass spectrometry (GC/MS). There was a six-fold enrichment of 6KF_1α levels in isolated microvessels, compared to homogenates, suggesting that 6KF_1α is of vascular, rather than neuronal origin. In order to further understand any possible role that norepinephrine (NE)_might have on modulation of PG synthesis, we studied the effects of 0.5 mM NE on PG synthesis from endogenous AA and from ³H-PGG₂, the endoperoxide precursor of PGs. In cat cortical homogenates NE induced a 74% increase in PGD₂ and PGF_2α, a 62% increase in PGE₂, and a 36% increase in 6KF_1α, as measured by GC/MS. NE caused a twofold increase in the conversion of ³H-PGG₂ to ³h-PGG_2α, with a concomitant decrease in ³H-PGE₂ and ³H-6KF_1α formation, and no change in ³H-PGD₂ synthesis. NE had no effect on the total conversiob of ³H-PGG₂ to ³H-PGs, nor on the breakdown of ³H-PGG₂ in the absence of brain tissue. We conclude that NE stimulates extravascular synthesis of PGD₂, PGE₂ and PGF_2α by stimulation of the prostaglandin synthetase complex, in addition to NE's stimulatory effect on the conversion of PGG₂ to PGF_2α, and that the lack of effect of NE on 6KF_1α synthesis reflects either a failure to achieve an adequate concentration at the vascular tissue, or an absence of the mechanism whereby NE stimulates PG synthetase.     

A study of the ability of H-2Kk-Iak containing subcellular fractions to elicit primary anti-H-2 cytotoxic T lymphocytes

In order to identify an antigen required for elicitation of anti-H-2 cytotoxic T lymphocytes (CTLs), we have purified the H-2-K^k glycoprotein, incorporated it into a lipid vesicle, and tested it for its ability to elicit anti-H-2K^k CTLs. The results indicate that a lipid vesicle containing purified H-2K^k can elicit specific secondary anti-H-2K^k CTLs. In addition it was shown that in association with a partially purified Ia^k fraction the primary anti-H-2K^k CTL response was enhanced. It was also shown that Ia^k antigens alone could elicit an anti-H-2^k CTL response. Although a low primary response was found with purified H-2K^k, it was observed that lipid vesicles containing both H-2K^k and Ia^k glycoproteins could elicit a significantly enhanced primary anti-H-2K^k CTL response. Lipid vesicles containing H-2K^k-Ia^k were tested for their enhanced capacity to elicit anti-H-2 CTLs as well as for their ability to elicit anti-H-2K^k CTLs in the presence of supernatants from concanavalin A-stimulated spleen cells.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Initiation of glycogen biosynthesis in Escherichia coli studies of the properties of the enzymes involved

The properties of the enzymes involved in the initiation of glycogen biosynthesis in Escherichia coli were studied.It was found that the enzymic activities which transfer the glycosyl residues from UDPglucose or ADPglucose for the glucoprotein synthesis had differing stabilities upon storage at 4°C.The small amount of glycogen and the saccharide firmly bound to the membrane preparation, were degraded during the storage period.The activity measured in fresh and in stored preparations gave different time dependence curves. The stored preparation had a lag period which could be due to the transfer of the first glucose units to the protein.Both UDPglucose and ADPglucose: protein glucosyltransferases were affected in different ways by detergents.Based on the results presented, it may be concluded that both enzymatic activities are due to different enzymes. Furthermore, both enzymatic activities are different from that which transfers glucose from ADPglucose to glycogen.The following mechanism for the de novo synthesis is suggested. Glycogen in E. coli could be initiated by two different enzymes which transfer glucose to a protein acceptor either from UDPglucose or ADPglucose. Once the saccharide linked to the protein has reached a certain size it is almost exclusively enlarged by another ADPglucose-dependent enzyme. The participation of branching enzyme will produce a polysaccharide with the characteristics of glycogen.     

A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH

The addition of bovine serum albumin (BSA) to 25 ± 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 ± 10 nm diameter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. alpha-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.