Photoactive amino acid derivatives with long alkyl chains

Summary Azobenzene amphiphiles containing-alanine, L-lysine or-homolysine moieties were synthesized and characterised. Stable monomolecular layers at the air/water interface and LB multilayers has been obtained from some of them. One sample of azobenzenes synthesized has been shown to undergo a reversible trans/cis photoisomerization upon light irradiation in both solution and LB multilayer.Abbreviations DCC N,N-dicyclohexyl carbodiimide - HONSu N-hydroxy succinimide - Boc t-butyloxycarbonyl - Bu^t t-butyl - EtOAc ethyl acetate - THF tetrahydrofuran - TEA triethylamine - HPLC high performance liquid chromatography - TLC thin layer chromatography - Ph phenyl - i-PrOH isopropyl alcohol     

Enzymatic synthesis of lactic acid derivatives with emulsifying properties

Novozym 435 (50 g l^–1) catalyzed the synthesis of n-octyl--d-glucopyranoside lactate by transesterification between n-octyl--d-glucopyranoside (30 g l^–1) and ethyl lactate (100 g l^–1) in acetone. In 12 h, a molar yield of 87% n-octyl -d-glucopiranoside lactate was obtained at a overall conversion of 90%.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

Reexpression of HPRT activity following cell fusion with polyethylene glycol

Polyethylene glycol-1000 (PEG-1000) induced fusion of HPRT (E.C. 2.4.2.8) deficient Chinese hamster cells with -galactosidase A (E.C. 2.3.1.22) deficient cells from a patient with Fabry's disease yielded hybrids which contained both human and hamster HPRT, G6PD (E.C. 1.1.1.49), and APRT (E.C. 2.4.2.7) and Chinese hamster -galactosidase B. Thus PEG-1000 mediated somatic cell fusion led to reexpression of Chinese hamster HPRT. It did not restore the expression of human -galactosidase. Since PEG-1000 treatment of HPRT^– Chinese hamster cells in the absence of human cells yielded no HPRT⁺ cells, it is concluded that the element responsible for the restoration of rodent HPRT was contributed by the human cells and not by the agent employed to promote fusion.This work was supported by research grants from the United States Public Health Services GM 17702, from the National Science Foundation BMS 74-21424, and from the National Foundation March of Dimes 1-377.     

Crystal structure of the pig pancreatic alpha-amylase complexed with malto-oligosaccharides

The structural X-ray map of a pig pancreatic -amylase crystal soaked (and flash-frozen) with a maltopentaose substrate showed a pattern of electron density corresponding to the binding of oligosaccharides at the active site and at three surface binding sites. The electron density region observed at the active site, filling subsites –3 through –1, was interpreted in terms of the process of enzyme-catalyzed hydrolysis undergone by maltopentaose. Because the expected conformational changes in the flexible loop that constitutes the surface edge of the active site were not observed, the movement of the loop may depend on aglycone site being filled. The crystal structure was refined at 2.01 å resolution to an R factor of 17.0% (R _free factor of 19.8%). The final model consists of 3910 protein atoms, one calcium ion, two chloride ions, 103 oligosaccharide atoms, 761 atoms of water molecules, and 23 ethylene glycol atoms.     

A convenient method for the synthesis of norstatine derivatives from N-Fmoc or N-Boc amino acids

Summary A new and convenient method for synthesizing norstatine derivatives has been developed. The key step is the preparation of propargylic alcohols from N-Fmoc or N-Boc amino aldehydes, followed by transformation to carboxylic acids after protection of the alcohols. The N-Boc-protected derivatives are coupled to a dipeptide sequence to afford peptide mimetics (norstatines).     

Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: Effects of substance P-receptor inhibition

Dietary deficiency of magnesium (Mg) in rodents results in cardiomyopathic lesion formation. In our rat model, these lesions develop after 3 weeks on the Mg-deficient diet; significant elevation of several cytokines, IL-1, IL-6 and TNF also occurs. In probing the mechanisms of lesion formation, we obtained data supporting the participation of free radicals (Freedman AMet al.: Bioch Biophys Res Commun 1990; 170: 1102). Recently, we identified an early elevation of circulating substance P and proposed a role of neurogenic peptides during Mg-deficiency (Weglicki WB, Phillips TM: Am J Phys 1992; 262: R734). The present study was designed to evaluate the contribution of neurogenic peptides to the pathogenesis of Mg-deficiency. In the blood, substance-P and calcitonin gene related peptide (CGRP) are elevated during the first week on the diet. During the second week, circulating histamine, PGE₂ and TBAR-materials were elevated and red cell glutathione was reduced, all prior to the elevation of the inflammatory cytokines during the third week. When the rats were treated with the substance P-receptor blocker [CP-96,345], the levels of substance P and CGRP remained elevated; however, increases in histamine, PGE₂, TBAR-materials, and the decrease in red cell glutathione were inhibited; also, the development of cardiac lesions was inhibited significantly. These data support a central role for neurogenic peptides, especially substance P, in the development of cardiomyopathic lesions during Mg-deficiency.     

A fast and simplein situ method for the determination of the absolute configuration of amino acid esters using the chiroptical properties of their Eu(FOD)3 complexes

Summary The NMR shift reagent, Europium(III)-tris-(1,1,1,2,2,3,3)-heptafluoro-7,7-dimethyl-4-6-octanedione [Eu(fod)₃], complexes efficiently with-amino acid esters in chloroform. These complexes exhibit characteristic circular dichroism (CD) spectral patterns in the 350-250 nm region. A fast and simple procedure (also in microscale) has been worked out which utilizes the signs of these CD bands for the determination of the absolute configuration at the-carbon atomin situ. In the L-series, a positive CD band is observed at around 310 nm and a negative one in the 290-280 nm region. The CD spectra of the Eu complexes of the D-isomers are mirror images of those of the L-configurations. An empirical rule is proposed.Presented in part at the 2nd International Congress on Amino Acids and Analogues, Vienna, Austria, August 5–9, 1991.     

Synthesis of peptides employing Fmoc-/Boc-/Z-amino acid fluorides and activated commercial zinc dust

The coupling of urethane protected amino acidfluorides is accomplished in the presence ofactivated, commercial zinc dust to synthesize severaldi- and tripeptides.The coupling was fast and racemization free. The yieldas well as purity of the peptides was satisfactory.The method was extended for the incorporation ofsterically hindered ,-dialkylaminoacids and N-methylamino acids as well.     

Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

Synthesis of aminobenzyltriethylenetetraaminohexaacetic acid: conjugation of the chelator to protein by an alkylamine linkage

The conjugation of a chelating agent to an antibody as an anchoring site for a radionuclide is the first step in the successful preparation of a radiolabeled antibody for a diagnostic and therapeutic application. The high affinity of the protein bound chelator towards radionuclide ensures a higher selectivity in the delivery of the radionuclide to the targeted tissue. 4-Aminobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentate chelating agent has been now prepared for conjugation with proteins in view of the higher affinity of TTHA metal ions as compared to DTPA. The latent crosslinking potential of -hydroxy aldehydes has been used to conjugate the new chelating agent to proteins through an alkylamine linkage. On incubation of amino benzyl TTHA with glycoladehyde at neutral pH and room temperature, the reagent is converted to oxoethyl amino benzyl TTHA. On addition of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA generates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by sodium cyanoborohydride stabilizes the schiff base adducts as stable alkylamine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugated to albumin through a thiocarbamoyl linkage. Both preparations of TTHA conjugated albumin complexed with ^99mTc and ¹¹¹In, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after the preparation. The radiolabels complexed with these TTHA -albumin conjugates could not be chased out by free DTPA. A comparison of the biodistribution of ¹¹¹In, bound to the TTHA conjugated through an alkylamine and a thiocarbamoyl linkage showed that ¹¹¹In complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen with thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, the alkylamine linkage appears to be more stable under the in vivo conditions. The glycolaldehyde mediated alkylation procedure offers a mild, simple and rapid method for preparation of drug-protein (antibody) conjugates.     

Lipopolysaccharide (LPS) increases thein vivo oxidation of branched-chain amino acids in the rat: A cytokine-mediated effect

Septic rats (as induced by cecal puncture and ligation) showed an increased rate ofin vivo leucine oxidation as measured from the formation of¹⁴CO₂ from an intravenously injected [1-¹⁴C]leucine tracer dose. Acute lipopolysaccharide (LPS) administration (1 mg/kg) to rats caused a similar effect on the rate ofin vivo leucine oxidation. Additionally, both tumour necrosis factor- (TNF) and interleukin-1- (IL-1), in an acute dose of 100 g/kg, also increased the rate of the oxidation of the amino acid, although only IL-1 caused a similar increase to that observed following LPS. The observed increased leucine oxidation was related to lower leucine concentrations both in LPS- and cytokine-treated rats. Important decreases were also observed in the other branched-chain amino acids (valine and isoleucine) in the LPS- and IL-1-treated animals. Isolated incubated muscles from TNF- and IL-1-treated rats did not show any changes in the rate of leucine utilization, thus suggesting that the mechanism by which the cytokines stimulate whole-body leucine oxidation is not based on an increase in the activity of the enzymatic machinery responsible for leucine oxidation. Additionally, glucocorticoids do not seem to mediate the enhancedin vivo oxidation of the amino acid since, although they are increased by both LPS and cytokines, treatment of the animals with RU486 (a glucocorticoid antagonist) was not able to suppress the effects of the cytokine onin vivo leucine oxidation.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Saccharomyces cerevisiae DNA repair processes: an update

The effect of incubating T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly , and isoforms) was assessed by immunoblotting, enzyme activity assay and [³H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC and PKC had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca²⁺) indicated that translocation of PKC and was very rapid — detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight (authentic) PKC immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC moved more rapidly than PKC which appeared to translocate more quickly than PKC . After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC was not translocated by LHRH. (Mol Cell Biochem 165: 65–75, 1996)     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

Interaction of Duodenase with Serpins from Human Blood Serum

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, ₁-protease inhibitor (₁-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for ₁-PI and ACT, respectively. The presence of a stable enzyme–inhibitory complex duodenase–₁-PI was confirmed by SDS-PAGE. The formation of the duodenase–ACT complex was not demonstrated; instead, the band of the cleaved inhibitor indicated the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (k × 10⁵, ^–1 s^–1) were 2.4 ± 0.3 × 10⁵ for ₁-PI and 3.0 ± 0.4 × 10⁵ for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: a consequence of the formation of "native-like conformation".

The influence of n-propanol on the overall -helical conformation of -globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of -helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of -helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of -globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The -helical conformation induced into - and -globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the -helical conformation of both - and -chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced -helical conformation of globins and the -helical conformation of - and -chains. A cross-correlation of the n-propanol induced increase in the fluorescence of -globin with the corresponding increase in the -helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the -helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a native-like structure. The induction of -helical conformation into these proteins in the presence of n-propanol and the consequent generation of native-like conformation is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an -helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume -helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high -helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of -helical proteins. Consistent with this concept, the induction of -helical conformation into shorter polypeptide fragments of 30 residues, (e.g., _1-30, which exists in an -helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.