Gas chromatographic determination of total amino acids and protein in sediments using the ninhydrin-CO2 reaction

A sensitive modification of the ninhydrin-CO₂ method involving the gas chromatographic determination of the total protein and amino acid content of sediment is described. The method gives a linear response over the amino acid concentration range 10^?5 M to 4 × 10^?2 M. It can be used for whole sediment, hydrolysates and interstitial water. The performance of the method is compared with the fluorescamine method for primary amines.     

Methodology for assessing respiration and cellular incorporation of radiolabeled substrates by soil microbial communities

A method is described for determining biodegradation kinetics of both naturally occurring and xenobiotic compounds in surface and sub-surface soil samples. The method measures both respiration and uptake into cellular biomass of¹⁴C-labeled substrates. The estimation of biomass incorporation entailed removal of cells from soil particles by washing the soil with a polyvinyl-pyrrolidone/pyrophosphate solution and H₂O₂. After separation of the cells and the soil particles by centrifugation, the cells were trapped on membrane filters for liquid scintillation counting. Mass balances were easily obtained. The technique was used to measure metabolic activity in soil profiles, including unsaturated and saturated zones. First order rate constants (K₁) were in the range of 10^–3–10^–2 hour^–1 for amino acid metabolism and 10^–5–10^–4 hour^–1 for m-cresol metabolism. Saturation kinetics were observed for amino acids and m-cresol. m-Cresol K₁ values for uptake often exceeded those for respiration by greater than a factor of ten. V_max values were low (amino acids, 10¹–10² ng g^–1 hour^–1; m-cresol, 10^–1 ng g^–1 hour^–1), whereas K_m values were quite high (amino acids, 10³–10⁴ ng g^–1; m-cresol 10³–10⁵ ng g^–1). Saturation was not observed in many horizons even at 10⁵ ng g^–1 dry soil. Frequently, respiration obeyed saturation kinetics whereas uptake was first order. It is concluded that measuring only kinetics of respiration may lead to severe underestimations of biodegradation rates.     

The uptake and incorporation of leucine and cystine by the mycelial and yeastlike phases of Blastomyces dermatitidis

Uptake and incorporation of L-leucine-C¹⁴ and L-cystine-S³⁵ was studied in the mycelial [MP] and yeastlike [YP] phases of the dimorphic fungal pathogen,Blastomyces dermatitidis. Both amino acids entered the cells of the two morphological forms ofB. dermatitidis by a permease-like system at low external concentrations of substrate. At high substrate levels, the amino acids entered the cells by a simple diffusion-like process in addition to the permease-like system. Michaelis-Menten constants [K_m] for L-leucine was found to be 1.1×10^–5 M and 4.4×10^–5 M for the MP and YP phases, respectively. The K_m for L-cystine was found to be 1.0×10^–5 M for the MP and 0.5×10^–5 M for the YP. A requirement for energy supplied by metabolic activity was demonstrated by the inhibition of uptake and incorporation of the amino acids by cells incubated with either 2,4-dinitrophenol or sodium azide. Amino acid uptake was broadly tolerant of hydrogen ion concentration, but definite optima were demonstrated at pH 7.0 to 7.5.     

Organic nitrogen stimulates caulogenesis from hypocotyl callus of Phyllanthus fraternus

Factors involved in promoting caulogenesis from hypocotyl explants of Phyllanthus fraternus were studied. Hypocotyl explants were cultured on B₅ medium supplemented with 2,4-D or NAA in the presence and absence of BAP (at concentrations 0, 10^–7, 10^–6 and 10^–5M). Adventitious shoots differentiated from callus developed from the cut ends of 12.5% of the hypocotyl segments cultured on medium supplemented with 10^–6M BAP in combination with 10^–6M 2,4-D or 10^–6M NAA. Profuse rooting occurred from the hypocotyl explants on medium supplemented with 10^–6M BAP + 10^–6M NAA. Incorporation of casein hydrolysate in B₅ medium along with 10^–6M BAP + 10^–7M 2,4-D enhanced the frequency of cultures with adventitious shoots upto 68.0%. Glutamine, glutamic acid or proline could partially substitute for the effect of casein hydrolysate. Amongst the hypocotyls from 3–14 d old seedlings, the best caulogenesis was obtained with hypocotyls from 7 d old seedlings both in presence or absence of casein hydrolysate. Best rooting of shoots was achieved on half-strength B₅ medium supplemented with 10^–6M IBA. After hardening, plantlets were successfully transferred to the soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2, 4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - CH casein hydrolysate - Arg L-arginine - Glu L-glutamic acid - Gln L-glutamine - Leu L-leucine - Lys L-lysine - Pro L-proline     

Nitrogen fixation in subarctic streams influenced by beaver (Castor canadensis)

Nitrogen fixation was measured in four subarctic streams substantially modified by beaver (Castor canadensis) in Quebec. Acetylene-ethylene (C₂H₂ C₂H₄) reduction techniques were used during the 1982 ice-free period (May–October) to estimate nitrogen fixation by microorganisms colonizing wood and sediment. Mean seasonal fixation rates were low and patchy, ranging from zero to 2.3 × 10^–3 µmol C₂H₄ · cm^–2 · h^–1 for wood, and from zero to 7.0 × 10^–3 µmol C₂H₄ · g AFDM^–1 · h^–1 for sediment; 77% of all wood and 63% of all sediment measurements showed no C₂H₂ reduction. Nonparametric statistical tests were unable to show a significant difference (p > 0.05) in C₂H₂ reduction rates between or within sites for wood species or by sediment depth.Nitrogen contributed by microorganisms colonizing wood in riffles of beaver influenced watersheds was small (e.g., 0.207 g N · m^–2 · y^–1) but greater than that for wood in beaver ponds (e.g., 0.008 g N · m^–2 · y^–1) or for streams without beaver (e.g., 0.003 g N · m^–2 · y^–1). Although mass specific nitrogen fixation rates did not change significantly as beaver transform riffles into ponds, the nitrogen fixed by organisms colonizing sediment in pond areas (e.g., 5.1 g N · m^–2 · y^–1) was greater than that in riffles (e.g., 0.42 g N · m^–2 · y^–1). The annual nitrogen contribution is proportional to the amount of sediment available for microbial colonization. We estimate that total nitrogen accumulation in sediment, per unit area, is enhanced 9 to 44 fold by beaver damming a section of stream.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Isolation: Analysis and Properties of Three Bradykinin–Potentiating Peptides (BPP-II, BPP-III, and BPP-V) From Bothrops Neuwiedi Venom

In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a K _i of the order of 9.7 × 10^–3 M to BPP-II, 7 × 10^–3 M to BPP-III, and 3.3 ×10^–3 M to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-lIle and 2Glu-2Gly-lSer-3Pro-2Val-Ille, with molecular weight of 1372, 1046, and 1078, respectively.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl₂, LaCl₃, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl₂ at concentrations above 10^–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10^–5 to 5×10^–5M) and LaCl₃ (7.5×10^–5 to 2.5×10^–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl₂, and LaCl₃ is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10^–4 to 10^–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl₃ nor nifedipine inhibit the formation of CaCl₂- or CTC-plugs. The unusual effects of these substances, which normally act as Ca²⁺ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La³⁺ mimicks the effects of calcium and that nifedipine binding to the Ca²⁺ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca²⁺.Abbreviations AFW artificial fresh water - CTC chlortetracycline - DCMU dichlorphenyldimethylurea - DMSO dimethylsulfoxide - EGTA ethyleneglycoltetraacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TAPS N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid     

Diffusive exchange of linear alkylbenze suifonates (LAS) between overlying water and bottom sediment

Linear alkylbenzenesulfonate (LAS) was detected in a 0–30 cm deep sediment column collected in Lake Teganuma (one of the most polluted lakes in Japan). The range of the LAS concentration in sediments was between 0.1 and 500 µg g^–1 (C₁₁-C₁₄ homologs per dry solid) and its vertical profile showed a seasonal variation. A mathematical model, which includes a diffusion term and a biodegradation term, was used to simulate the temporal variation of LAS in the sediment column and to calculate the diffusive flux rate of LAS across the sediment/water interface. An averaged diffusion coefficient of 2.4 × 10^–5 cm² s^–1 for the sediment interstitial water was obtained from sediment core samples located in Lake Teganuma. The biodegradation rate constant (0.002 d^–1) of LAS in the sediment obtained from the model analysis was considerably less than that reported for LAS in anaerobic waters. These results confirm that a model describing diffusive transport and biodegradation of LAS in the sediments can simulate the temporal variation of LAS in near surface sediments. The diffusive flux rate from overlying water to bottom sediment was calculated to be between –0.20 and 0.52 (C₁₁-C₁₄ LAS) mg m^–2 h^–1 and the annual net flux rate was 0.7 g m^–2 y^–1.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Stimulatory effect of hormonal signaling factors on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes: Cross talk with regucalcin

The effect of hormonal signaling factors on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10^–7–10^–5M), dibutyryl cAMP (10^–4 and 10^–3M) or inositol 1,4,5-trisphosphate (IP₃; 10^–6 and 10^–5 M) in the enzyme reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10^–4 M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5×10^–3 M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 M) with an effective concentration completely inhibited the effect of inositol-glycan (10^–5 M) to increase (Ca²⁺–Mg²⁺)-ATPase activity, while the effect of dibutyryl cAMP (10^–3M) or IP₃ (10^–5M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP₃. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca²⁺–Mg²⁺)-ATPase activity in liver plasma membranes.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Dehalogenation of 4-chlorobenzoate

Pseudomonas sp. CBS3 is capable of growing with 4-chlorobenzoate as sole source of carbon and energy. The removal of the chlorine of 4-chlorobenzoate is performed in the first degradation step by an enzyme system consisting of three proteins. A 4-halobenzoate-coenzyme A ligase activates 4-chlorobenzoate in a coenzyme A, ATP and Mg²⁺ dependent reaction to 4-chlorobenzoyl-coenzyme A. This thioester intermediate is dehalogenated by the 4-chlorobenzoyl-coenzyme A dehalogenase. Finally coenzyme A is split off by a 4-hydroxybenzoyl-CoA thioesterase to form 4-hydroxybenzoate. The involved 4-chlorobenzoyl-coenzyme A dehalogenase was purified to apparent homogeneity by a five-step purification procedure. The native enzyme had an apparent molecular mass of 120,000 and was composed of four identical polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.7. The maximal initial rate of catalysis was achieved at pH 10 at 60 °C. The apparent K _m value for 4-chlorobenzoyl-coenzyme A was 2.4–2.7 µM. V _max was 1.1 × 10^–7 M sec^–1 (2.2 µmol min^–1 mg^–1 of protein). The NH₂-terminal amino acid sequence was determined. All 4-halobenzoyl-coenzyme A thioesters, except 4-fluorobenzoyl-coenzyme A, were dehalogenated by the 4-chlorobenzoyl-CoA dehalogenase.Abbreviations CBA chlorobenzoate - CoA coenzyme A - HBA hydroxybenzoate - DTT dithiothreitol - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis     

Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

We have used a novel spectrophotometric method to study the hydrolysis of N-acetylphenylalanyl adenylate anhydride (AcPhe-AMP) and phenylalanyl-adenylate anhydride (Phe-AMP) at low concentrations (10^–5 M), 25 °C, constant buffer concentration (0.05 M), and as a function of pH. While Phe-AMP is susceptible principally to attack by OH^–, with two different rates depending on whether the -amino group of the amino acid is protonated or not, the AcPhe-AMP is susceptible to acid decomposition as well. At pH's 4–8, the Phe-AMP hydrolyzes faster than the AcPhe-AMP, but at pH less than 4 or pH greater than 8, the blocked form hydrolyzes faster. Both forms are also attacked by H₂O, and at the same rate. Moreover, the hydrolysis of Phe-AMP is shown to be greatly catalyzed by carbonate, although the AcPhe-AMP is not subject to such catalysis. The rate laws for the various mechanisms and the activation energies for the hydrolyses at pH 7.1 are given.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.