Hydrophobic channels in crystals of an α-aminoisobutyric acid pentapeptide

The crystal structure of the pentapeptide p-toluene-sulfonyl-(α-aminoisobutyryl)₅-methyl ester (Tosyl-(Aib)₅-OMe) has been determined in the space group P$\text{I}$. Pentapeptide molecules are folded in the 3₁₀ helical conformation and packed together, so as to yield a hydrophobic channel with a minimim diameter of 5.2 Å. The channel contains crystallographically disordered material. This structure provides a model for channel formation by hydrophobic peptide aggregates and should prove useful in studies of alamethicin, suzukacillin and related Aib containing membrane channels. Triclinic (P$\text{I}$) crystals of the pentapeptide are obtained in the presence of LiClO₄ in aqueous methanol, whereas crystallization from methanol alone yields crystals in the space group Pbca. The conformations of the peptide in the two crystal forms are very similar and only the molecular packing is dramatically different.     

Diglyceride/monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation.     

Characterization of two enzyme proteins catalyzing NADP+/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 from swine kidney

Two proteins (pI 4.8 and 5.8) capable of catalyzing NADP⁺/NADPH-dependent oxidoreduction of prostaglandins at C-9 and C-15 but not at C-11 have been purified to homogeneity from swine kidney. Both proteins exhibited identical molecular weight and subunit size. Similar amino acid composition, antigenic determinants, and coenzyme and substrate specificity were also found. The molecular weight of the enzyme as determined by gel filtration was 29,000. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel gave a value of 29,500 indicating the presence of a single polypeptide chain. Either enzyme protein utilized a variety of prostaglandins (PGS) as substrates. PGA₁-glutathione conjugate and PGB₁ were found to be the best substrates for prostaglandin 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase activities, respectively. For prostaglandins having dual reactive groups in a single molecule, the rate of oxidation of PGF_2α at C-15 was comparable to that at C-9, whereas the rate of reduction of 15-keto-PGE₂ at C-15 was far greater than that at C-9.     

Stimulation of cyclic nucleotide phosphodiesterase by products of phosphatidylinositol metabolism catalyzed by phospholipase A2

Products of phospholipase A₂-catalyzed hydrolysis of phosphatidylinositol, lysophosphatidylinositol, and unsaturated fatty acid are very effective stimulators of a partially purified bovine brain cyclic nucleotide phosphodiesterase. More than 10-fold of calcium-independent stimulation can be achived by lysophatidylinositol or oleic acid. The degree of stimulation is comparable to that by calcium-mediated calmodulin. The other lysophospholipid which shows comparable stimulation is lysophosphatidylserine although at a higher concentration. Diacylphosphoglycerides are inactive. Kinetic studies showed that lysophosphatidylinositol, like calmodulin, increased the V_max without affecting the apparent K_m for adenosine 3′,5′-cyclic phosphate (cAMP).     

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.     

Studies on the degradation of human very low density lipoproteins by human milk lipoprotein lipase

Human milk lipoprotein lipase (LPL) was purified by heparin-Sepharose 4B affinity chromatography. The time required for the purification was approximately 2 h. The acetone-diethyl ether powder of milk cream was extracted by a 0.1% Triton X-100 buffer solution and the extract was applied to the heparin-Sepharose 4B column. The partially purified LPL eluted by heparin had a specific activity of 5120 units/mg which represented a 2500-fold purification of the enzyme. The LPL was found to be stable in the heparin solution for at least 2 days at 4 °C. This enzyme preparation was found to be free of the bile salt-activated lipase activity, esterase activity, and cholesterol esterase activity. The LPL had no demonstrable basal activity with emulsified triolein in the absence of a serum cofactor. The enzyme was activated by serum and by apolipoprotein C-II. The application of milk LPL to studies on the in vitro degradation of human very low density lipoproteins can result in a 90–97% triglyceride hydrolysis. The LPL degraded very low density lipoprotein triglyceride and phospholipid without any effect on cholesterol esters. Of the partial glycerides potentially generated by lipolysis with milk LPL, only monoglycerides were present in measurable amounts after 60 min of lipolysis. These results show that the partially purified human milk LPL with its high specific activity and ease of purification represents a very suitable enzyme preparation for studying the kinetics and reaction mechanisms involved in the lipolytic degradation of human triglyceride-rich lipoproteins.     

Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypanosoma brucei requires Ca2+

A-antigenicity; Glycocalyx; Hydrolases; Membrane glycoproteins     

Release of ascorbate from a synaptosomal fraction of rat brain

We have studied factors controlling the release of endogenous ascorbate from synaptosomes prepared from various regions of the rat brain. Ascorbate was spontaneously released from synaptosomes, and this efflux could be enhanced by incubation at 37°C. A further additional ascorbate release could be induced by potassium depolarization or, in striatal, hippocampal and cortical synaptosomes, by incubation with the amino acid glutamate. Spontaneous, depolarization and glutamate-evoked ascorbate release were shown to occur by separate mechanisms. Glutamate-evoked ascorbate release occurred by a heteroexchange mechanism. In cerebellar synaptosomes there was no evidence for such heteroexchange; however, in synaptosomes of this brain region kainic acid induced ascorbate release, probably by acting on excitatory amino acid receptors. The results are discussed in relation to the changes in extracellular brain ascorbate occurring in vivo.     

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.     

Mechanism of action of the nitrosoureas: formation of 1,2-(diguanosin-7-yl) ethane from the reaction of BCNU (1,3-bis-[2-chloroethyl]-1-nitrosourea) with guanosine

A crosslinked dinucleoside, 1,2-(diguanosin-7-yl) ethane, has been isolated from the reaction of guanosine with the antitumor agent, BCNU. The formation of this product suggests that DNA crosslinking, which may be responsible for the cytotoxicity of BCNU, could occur through such dinucleosides.     

Comparative study of the iron-binding properties of human transferrins: I. Complete and sequential iron saturation and desaturation of the lactotransferrin

Human lactotransferrin binds 2 Fe³⁺ tightly at two specific sites. In order to demonstrate differences between the stability of the two iron-binding sites, the removal of iron was studied in buffers in the pH range 8-3 varying the ionic strength and with or without metal chelators such as phosphate ions and EDTA.The results show that in the presence of formate and acetate buffers of ionic strength 0.1–0.4 and in a pH range of 5–3, the two Fe³⁺ from human lactotransferrin are removed stimultaneously.Addition of 4 mM EDTA to buffers of ionic strength 0.1 and in the pH range 8–3 shows that between pH 5–4.3 the iron from only one of the binding sites, called the ‘acid labile’ site, is removed.Addition of 0.2 M phosphate ions to buffers of ionic strength 0.2 and in pH range 8–3 containing 4 mM EDTA shows that Fe³⁺ from the ‘acid labile’ site may be completely removed at pH 6. Removal of Fe³⁺ from the ‘acid stable’ site is obtained at pH 4.The differential behavior of the two iron binding sites was also shown by saturation experiments in the presence of citrate/bicarbonate buffers at different pH values. In a pH range 6.2–4.8, 50% saturation was obtained, but at pH 6.35 complete saturation was achieved. When saturation of partially saturated samples of human lactotransferrin was performed with ⁵⁹Fe it was demonstrated that in the pH range 6.2–4.8 iron is bound only to the ‘acid labile’ site.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

Isolation and characterization of a translation inhibitor from human term placenta

An inhibitor of protein synthesis has been isolated from free cytoplasmic ribonucleoprotein particles of human term placenta. The inhibitor is resistant to phenol, DNase, proteinase K, and heating at 100 degrees C, but is sensitive to alkaline hydrolysis. These data suggest that the inhibitor is RNA. Experiments provide evidence that this preparation contains no RNase contaminant and does not induce an RNase in this assay system. Three lines of evidence suggest that the inhibitor acts at the initiation of protein synthesis in the wheat germ translation system. First, a lag occurs before cessation of translation when the inhibitor is added to translating polyribosomes. This lag is identical to that seen upon the addition of aurintricarboxylic acid, a known inhibitor of initiation. Second, sucrose gradient analyses demonstrate that, when the inhibitor is present at the start of translation, 40 S complexes form, but neither 80 S complexes nor polyribosomes are seen. Third, gradient analyses show that, when the inhibitor is added to translating polyribosomes, 40 S complexes accumulate with a progressive loss of polyribosomes. Finally, the extent of inhibition depends upon the amount of wheat germ extract added to the reaction mixture and not the amount of mRNA present. This suggests an interaction between the inhibitor and a component of the wheat germ extract.     

The synthesis of glycopeptide fragments of human plasma alpha1-acid glycoproteins by sequential elongation at the terminal-amino group.

Glycopeptides corresponding to sequences 27--28, 48--49, and 58--59 of human plasma alpha1-acid glycoproteins have been synthesized by sequential elongation of the peptide chain at the terminal amino group. 2-Acetamido-3,4,6-tri-O-acetyl-1-N-(L-aspart-4-oyl)-2-deoxy-beta-D-glucopyranosylamine was condensed with the p-nitrophenyl esters of protected amino acids to give the corresponding protected glycodipeptides having the sequences Gly-(GlcNAc-4-)Asn, Pro-(GlcNAc-4-)Asn, Val-(GlcNAc-4-)Asn, Leu-(GlcNAc-4-)Asn, Glu-(GlcNAc-4-)Asn, Tyr-(GlcNAc-4-)Asn, Ser-(GlcNAc-4-)Asn, and Cys-(GlcNAc-4-)Asn. Deprotection of the carbohydrate and of the peptide residues of these compounds was achieved, except for those having N-tert-butyloxycarbonyl protective groups, to give the corresponding free glycopeptides. The glycotripeptide 2-acetamido-1-N-(2-N-[N-(tert-butyloxycarbonyl)-L-glutam-1-oyl-L-tyrosyl]-L-aspart-4-oxy)-2-deoxy-beta-D-glucopyranosylamine, having the amino acid sequence 10--12 of human plasma alpha1-acid glycoprotein, was prepared by condensation of 2-acetamido 3,4,6-tri-O-acetyl-2-deoxy-1-N-[2-N-(L-tyrosyl)-L-aspart-4-oyl[-beta-D-glucopyranosylamine with 5-benzyl 1-p-nitrophenyl N-(tert-butyloxycarbonyl)-L-glutamate, followed by removal of the ester groups.     

Purification of guanylate cyclase from human platelets and effect of arachidonic acid peroxide.

Guanylate cyclase of human platelets was separated from cyclic nucleotide and GTP hydrolytic activities with a 104-fold purification over the homogenate. The purified guanylate cyclase preparation requires neither the GTP regenerating system nor cyclic GMP but is stimulated by about 2-fold by 2.5 mM cyclic GMP. The molecular weight of the enzyme was estimated as 180,000 and the Km value for GTP was 95 μM. Arachidonic acid peroxide stimulated the purified enzyme by increasing maximum velocity without changing Km value.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Specificity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase, and nonspecific carboxylesterase from rat liver microsomes

The comparative substrate specificities of five purified serine hydrolases from rat liver microsomes have been investigated, especially their action upon natural lipoids. All enzymes had high carboxylesterase activities with simple aliphatic and aromatic esters and thioesters. The broad pH optima were in the range of pH 6-10. Synthetic amides were less potent substrates. The hydrolytic activities towards palmitoyl-CoA and monoacyl glycerols were generally high, whereas phospholipids and palmitoyl carnitine were cleaved at moderate rates. Acetyl-CoA, acetyl carnitine, and ceramides were not cleaved at all. The closely related hydrolases with the highest isoelectric points (pI 6.2 and 6.4) were most active with palmitoyl-CoA and palmitoyl glycerol. One of these enzymes might also be responsible for the low cholesterol oleate-hydrolyzing capacity of rat liver microsomes. Among the other hydrolases, that with pI 6.0 showed significant activities with simple butyric acid esters, 1-octanoyl glycerol, and octanoylamide. The esterase with pI 5.6 had the relatively highest activities with palmitoyl carnitine and lysophospholipids. The purified enzyme with pI 5.2 showed some features of the esterase pI 5.6, but generally had lower specific activities, except with 4-nitrophenyl acetate. The lipoid substrates competitively inhibited the arylesterase activity of the enzymes. The varying activities of the individual hydrolases were influenced in parallel by a variety of inhibitors, indicating that the purified hydrolases possessed a relatively broad specificity and were not mixtures of more specific enzymes. The nomenclature of the purified hydrolases is discussed.     

Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload

The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.