The thermal stability of bleaching intermediates and the metarhodopsins I in equilibrium II equilibrium have been examined for purified rhodopsin in cholic acid and a series of alkyl sucrose esters. The results are compared with rhodopsin contained in its natural phospholipid environment. It is found that detergents modify the transition temperatures of the photointermediates in a complex manner. The equilibrium between metarhodopsins I and II is also strongly dependent on the detergent species. It is shifted according to the micellar rigidity. This effect is reversible and allows direct observation of a back reaction from metarhodopsin II to I.
Study of the lymphokine interleukin 2 (IL2) began with several diverse qualitative observations in the middle 1970s. Rationalization of early work led to a focused program of research involving a number of laboratories. This recently culminated in the molecular description of both murine and human IL2. The glycoprotein, containing about 16 000 daltons of protein, is secreted in response to activators of T helper lymphocytes. A number of complex immune responses is stimulated by pure, recombinant IL2. The induction of IL2 and other lymphokines is apparently specific, in that only a small subset of proteins shows altered expression in the response. Moreover, the immunosuppressive agent cyclosporin A specifically inhibits the induction of mRNA for IL2 and certain other lymphokines, without affecting the expression of the majority of other proteins. Understanding of IL2 and its biological context has recently been enhanced by an intense activity on the part of biotechnology companies.
The divalent cations Mg2+ and Mn2+ in excess of the concentrations required to complex with ATP (excess Mg2+ or Mn2+) modulate the activity of adenylate cyclase. As a substrate, Mn X ATP was at least as effective as Mg X ATP in supporting adenylate cyclase activity in white and brown adipose tissue membranes. Both excess Mg2+ and Mn2+ had quantitatively different effects on the enzyme of lean and ob/ob mice and qualitatively different effects in white and brown adipose tissue. In white adipocyte membranes excess Mg2+ increase basal activity, as well as activity owing to guanylylimidodiphosphate (Gpp(NH)p) (with or without isoproterenol) and NaF. Maximal activation by Gpp(NH)p + isoproterenol required a higher concentration of Mg2+ in tissue from ob/ob than lean mice. Excess Mn2+ prevented the activation of the enzyme by Gpp(NH)p or Gpp(NH)p + isoproterenol in a dose-dependent manner. Mn2+ inhibited even in the presence of maximally effective Mg2+ concentrations. The enzyme of the ob/ob mouse membrane required a significantly higher dose of Mn2+ to achieve 50% inhibition. In brown adipose tissue, specific activities of the isoproterenol + Gpp(NH)p and NaF stimulated enzyme were significantly lower in the obese mice under all conditions studied. Except that NaF-stimulated activity was increased significantly more in the membranes of lean mice by the combination of Mg2+ + Mn2+, these cations did not produce significantly different dose-dependent effects in membranes from lean and ob/ob mice. Maximal activation occurred at lower concentrations MgCl2 (3-5 versus 10-20 mM) and required higher concentrations of MnCl2 (3-5 versus 1 mM) in brown than in white adipose tissue membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA--Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9-2.4% of the amount of protein applied to the column. They contained 20 +/- 1.3% hexose and 1.7 +/- 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate--polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50-55, 75, 95, 130, and 155 kdaltons along with minor bands of 20-40, 42, 45, 60-65, 175, and 200-250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 10(6) cells/mL in the presence of PHA. A 50% inhibition was observed when 20 micrograms/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 microgram/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent studies from our laboratory and others have shown that Km values for steroid substrates of the 3 beta-hydroxysteroid dehydrogenase in the human placenta were in the nanomolar range compared with micromolar values previously described. The purpose of the present study was to measure the kinetic parameters of the 3 beta-hydroxysteroid dehydrogenase in other human tissues, namely the ovary and chorion laeve, and to determine whether they were similar to those of the placental enzyme. In chorion laeve microsomes the 3 beta-hydroxysteroid dehydrogenase had Km values for dehydroepiandrosterone and pregnenolone similar to those found in placenta. Microsomes from human ovaries, on the other hand, had Km values for both substrates 10- to 20-fold higher. However, the ability of various steroids to inhibit the ovarian enzyme was similar to that previously described from the placenta and the chorion laeve.
The glutamylation of methotrexate catalyzed by beef liver folypolyglutamate synthetase (FPGS) is activated by addition of pteroic acid. Pteroic acid causes greater stimulation of FPGS, including glutamylation of tetrahydrofolate, at neutral pH values (i.e., below the pH optimum of 8.4). We have attributed this activation to a conformational change of FPGS induced by pteroic acid.
This work presents a quantitative study of the modulation of platelet responsiveness to sodium arachidonate by serum albumin. Rabbit platelets suspended in protein-free buffer containing dextran aggregate reversibly in response to micromolar amounts of ADP and sodium arachidonate. The optimal concentration of arachidonate for aggregation response is 5 microM. Inhibition occurs at higher concentrations and is not related to thromboxane A2 formation since arachidonate inhibits ADP-induced aggregation of aspirin-treated platelets. Thin-layer chromatographic studies show that, at the high arachidonate levels sufficient to almost completely abolish platelet aggregation, the synthesis of thromboxane A2 persists. Albumin relieves the inhibition caused by excess arachidonate, whether the stimulus is arachidonate itself or ADP. This effect is due to arachidonate binding and is optimal at fatty acid/protein ratios near 4; no stimulation of platelets was observed at ratios less than 2. The optimal concentration of arachidonate for stimulation of platelet aggregation occurs in the range where albumin buffers the free arachidonate concentration most effectively, hence the extremely narrow range of total arachidonate concentrations effective for platelet response seen in the absence of albumin is enormously broadened in the presence of albumin. Albumin inhibits conversion of arachidonate to thromboxane A2 and hydroxy acids, especially at ratios of arachidonate/albumin below 10. Bilirubin (an albumin ligand) has no effect on albumin modulation of platelet response until the bilirubin/protein ratio exceeds 2. Palmitate progressively displaces arachidonate from albumin and affects the range of effective arachidonate concentrations but not the maximum response.
After periods of 5 and 30 min following decapitation, rat cerebral cortices were removed and subcellular fractions were prepared. Fractions P1A (large myelin), P1B (nuclei), P1C (cells and debris), P2A (small myelin), P2B (synaptosomes), P2C (mitochondria), and P3 (microsomes) were isolated. Free fatty acid levels of 1.0 and 1.4 mumol/g tissue were found in the homogenates at the early and late times of ischemia. In the 30-min samples, P1A, P1C, and P2A had relatively high specific contents of total free fatty acids in comparison with other subfractions. At this time P2C was relatively enriched in arachidonate, P1A and P2A were enriched in palmitate, and P2B and P3 were enriched in stearate in comparison with the homogenate. P2C had the highest ratio of polyunsaturates/saturates in its free fatty acid pool. Comparing the 5- and 30-min samples, a large increase in the quantity of free fatty acids was found in fractions P1A and P2A, so that at the later time P1A + P2A contained 60 mol% of the free fatty acid in the total subfractions derived from cerebral cortex. In comparison with the homogenate, the lack of accumulation of free fatty acids in certain membranes known to possess phospholipase activities (e.g., phospholipase A2 in P2C) and the buildup of free fatty acids in P1A and P2A led to the hypothesis that free fatty acids may be migrating outwards from intracellular sites of production and accumulating in the multilamellar structure of myelin.
Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)
We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol-3-phosphate.
The number of actin genes per haploid genome of three sea star species has been measured by reassociation of a single-strand actin-coding sequence probe with excess genomic DNA. Least-squares fit analyses of the reassociation curve indicate five actin genes for Pisaster ochraceus and Pisaster brevispinus and eight actin genes for Dermasterias imbricata. Using 3′ untranslated sequence probes from cDNAs or 3′ noncoding regions of a genomic clone, essentially all of the actin-containing Southern digest fragments can be assigned to the three actin gene classes described earlier for P. ochraceus. Through analyses of genomic digests and dot blots from P. ochraceus probed with both the actin sequence and 3′ sequence, the copy number of gene class members per genome has also been estimated.
The effect of rat phosphorylcholine-binding protein (PCBP) and rabbit C-reactive protein (CRP) on the serum lipoprotein-heparin-Ca2+ precipitation reaction has been examined. Rabbit CRP, a nonglycosylated acute-phase protein, has been isolated from the serum after turpentine-induced inflammation following an isolation procedure similar to that used for rat PCBP. The mobilities of rabbit CRP and rat PCBP on polyacrylamide gel electrophoresis and immunoelectrophoresis have been found to be different. Rabbit CRP, even though a P-choline binding protein, was found to have no inhibitory effect on the serum lipoprotein-heparin-Ca2+ precipitation reaction. Desialylated rat PCBP obtained by enzymatic desialylation of rat PCBP no longer inhibited the precipitation reaction, even though it retained its P-choline binding property and immunochemical identity with rat PCBP. The results suggest that the binding of rat PCBP and rabbit CRP to the P-choline moiety is not a sufficient requirement, and it now appears that the sialic acid moiety must also be present on these proteins to observe the inhibition of lipoprotein precipitation. This requirement is fulfilled by rat PCBP, but not by rabbit CRP which is nonglycosylated.
3H-labelled insect juvenile hormone, which has previously been shown to stimulate the Na,K-ATPase of membrane preparations of the follicle cells of the ovary of Rhodnius prolixus, exhibits saturable binding to microsomal preparations from the follicle cells of vitellogenic and previtellogenic follicles. No saturable binding is detectable in preparations from brain. Scatchard analysis shows that there is a greater density of binding sites on the membranes from vitellogenic, as opposed to previtellogenic, follicles. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of solubilized membrane preparations shows that the hormone binds to peptides in two regions of the gel, corresponding to 43 000 and 150 000 daltons. [3H]Ouabain also binds to a peptide in the 150 000 region, as well as to the 100 000 region. Juvenile hormone thus may be binding directly to at least one of the constituent peptides of the Na,K-ATPase complex.
Microsomal fucosyltransferase isolated from sheep brain is strongly enhanced by charged lysophospholipids such as lysophosphatidylinositol and lysophosphatidic acid, while the corresponding phospholipids are inhibitive. Lysophosphatidylcholine (lyso-PC) also greatly increases the enzymatic activity and leads to its solubilization. Its stimulatory effect is related to the length of the fatty acyl chain involved in the lyso-PC structure: fatty acids C18 and C20 are less activating than the fatty acids C14–C16. Stimulation is restored when C18 fatty acids are unsaturated (e.g., C18:1–C18:3). Enzymatic activity enhancement is decreased when phosphatidylcholine structures are reformed by the addition of lyso-PC and the corresponding fatty acid. The physical state of these structures has no influence. These data provide evidence that bilayer structures do not modify enzymatic activity, while micellar structures formed by detergents and lysophospholipids lead to a strong increase in fucosyltransferase activity. However, lyso-PC does not interact in exactly the same way as Triton X-100. Although they both enhance the maximal velocity of fucosyltransferase for its two substrates, GDP-fucose and asialofetuin, the effect with lyso-PC is greater, and it clearly enables a better affinity for GDP-fucose. Endogenous phospholipids are also able to modify enzymatic activity. Hydrolysis of phosphatidylcholine by phospholipase A2 leads to an enzymatic stimulation.
A sensitive reverse-phase high pressure liquid chromatographic assay for formation of AMP coupled with analysis of aldehyde production has been used to characterize the properties of the fatty acid reductase complex of Photobacterium phosphoreum. The enzyme complex, which consists of three different polypeptides (34 000, 50 000, and 58 000), has a high affinity for ATP (Km = 20 nM) and shows highest specificity with C14 fatty acids. Activation of the fatty acid is efficiently coupled to the reduction step showing a stoichiometry of one molecule of fatty acid reduced to aldehyde and one molecule of NADPH oxidized for every molecule of ATP converted to AMP. Reconstituted fatty acid reductase (50 000 and 58 000) shows an ATP hydrolase activity that is independent of NADPH with the maximum amount of AMP formed limited by the amount of fatty acid in the assay, consistent with acyl-protein turnover experiments and the channeling of fatty acids to form acyl thioesters (−NADPH) or aldehyde (+NADPH). Addition of the 34 000 polypeptide to the reconstituted enzyme results in stimulation of AMP formation (−NADPH) to a level far exceeding the amount of fatty acid, showing that the fatty acid can be recycled by the 34 000 protein through its thioesterase activity. Also the 34 000 protein is responsible for a two- to three-fold stimulation in the rate of ATP hydrolysis, suggesting that it can be involved in the stabilization of the enzyme complex.
PS3 (thermophilic bacterium) cytochrome caa3 reacts slowly with cyanide which forms a low-spin complex with the CuBa3 centre. Partial reduction under catalytic conditions increases the rate of cyanide binding, and the reaction constant is rather similar to that of the mammalian enzyme, but the partially reduced complex dissociates more rapidly than does the corresponding eukaryotic complex. A simple biphasic reaction can account for the results obtained. The azide complex of partially reduced PS3 cytochrome caa3 shows an alpha-peak blue shift similar to that of the mammalian enzyme. PS3 cytochrome caa3 forms an oxyferri ("oxygenated") species like the mammalian enzyme, but does not undergo high- to low-spin changes during the aerobic steady state with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as substrates. Interactions seen in cytochrome oxidase-ligand reactions and its spin-state changes are therefore intrinsic to the enzyme's large catalytic subunits and do not require the presence of the small nuclear-encoded subunits found in eukaryotic systems.
Four proteolytic enzymes, euphorbains t 1 –t 4 , were isolated from the latex of the succulent Euphorbia tirucalli ("milk bush") and purified to homogeneity. The enzymes all have molecular weights of 74 000. Each enzyme has several different charged forms; t 1 , for example, has four with isoelectric points in the range of 5.0–5.5. The four proteases examined are of similar amino acid compositions but yield differing two-dimensional maps of tryptic digests. The euphorbain t's are not closely related in composition to their counterparts isolated from Euphorbia lathyris, Euphorbia pulcherrima, and Euphorbia cyparissias. The euphorbains from E. tirucalli were inhibited by phenylmethylsulphonyl fluoride and by histidine-specific reagents, and so are trypsin-like proteases.
Normal human erythrocytes were fractionated in a density gradient. Capacity to metabolize polyphosphoinositides was compared in young (least dense) and old (most dense) cells. Polyphosphoinositide synthesis was assessed by following the incorporation of radioactivity from [gamma-32P]ATP into the 1-(3-sn-phosphatidyl)-D-myo-inositol 4-phosphate (PtdIns4P) and 1-(3-sn-phosphatidyl)-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P2) of isolated membranes. There was no significant age-dependent change in the ability to synthesize PtdIns4P and PtdIns(4,5)P2 or in the response of the PtdIns and PtdIns4P kinases to Mg2+. The cytosolic Mg2+-dependent PtdIns(4,5)P2 phosphatase was also unaffected by age. The membrane cation-independent PtdIns4P phosphatase activity declined slightly (12%). Therefore, the capacity to catalyse the interconversion among the three phosphoinositides in the membrane is retained throughout the life of the erythrocyte. The Ca2+-dependent polyphosphoinositide phosphodiesterase activity in the membranes was reduced in old cells (57%) to the same extent as the glutamate-oxaloacetate transaminase activity used as an index of cell age. Thus, irreversible loss of polyphosphoinositide from the membrane by the action of this diesterase (prevented in healthy cells by the active maintenance of a very low intracellular Ca2+ concentration) is not very likely even in senescent cells when Ca2+ homeostasis begins to fail.
The structure of the core oligosaccharide of Vibrio ordalii has been investigated. The studies involved the use of nuclear magnetic resonance, methylation analysis, partial hydrolysis with hydrochloric acid, nitrous acid deamination, partial hydrolysis with sulfuric acid, Smith degradation, and oxidation with chromium trioxide. As a result of these studies the following structure is proposed.[Formula: see text]
The denaturations of ribonuclease A by calcium chloride and lithium chloride were studied by circular dichroism measurements in the far-ultraviolet region. The temperature dependence of the equilibrium constant for the unfolding of the protein by calcium chloride and lithium chloride gave values of 46 and 52 kcal mol-1 (1 cal = 4.1868 J) for the enthalpy of denaturation at 25 degrees C and pH 7.0, respectively. Thermodynamic parameters for the denaturation by calcium chloride and lithium chloride are compared with those for the heat and guanidine hydrochloride denaturation. It has been observed that the thermodynamic quantity, be it free energy, entropy, or enthalpy, cannot be related quantitatively to the extent of unfolding measured by various conformational properties of the protein.
