Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Synthesis of aspartame precursor by solid thermolysin in organic solvent

Summary The enzymatic synthesis of a peptide compound was carried out successfully in homogeneous organic solvent.Solid Thermolysin was found to catalyze the synthetic reaction of N-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester (Z-APM; a precursor of sweetner Aspartame) from N-benzyloxycarbonyl-L-aspartic acid (Z-L-Asp) and L-phenylalanine methyl ester (L-PheOMe) in a 98 percent organic medium (ethylacetatebenzenemethanolwater=5029192). The dissolution of enzyme was not observed. The optimal pH shifted to acidic side by 1.0 pH unit, compared with that in aqueous medium. The enzymatic activity of solid thermolysin with an average size of 3.4×9.5 m was determined to be 0.18 moles-product/(mg-solid)·h under the initial concentrations of L-PheOMe of 0.1M and Z-L-Asp of 0.05M, and at pH 6.0 and 40°C.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

The “interval timer”, photoperiod and temperature in the seasonal development of parthenogenetic and sexual morphs in the lime aphid,Eucallipterus tiliae L.

Summary The ability of the lime aphid to produce sexuals (males and females) as an alternative to parthenogenetic females is regulated by a timing mechanism (interval timer) which restrains the appearance of these morphs early in the year. In this species there are two interval timers: one controls the production of sexual females, and the other controls the production of males. Both intervals timers are sensitive to day-length and temperature but they respond in different ways.With the approach of autumn the waning effect of the interval timer inhibiting female production combined with the short day-lengths results in an increasing proportion of the aphids developing into sexual females. The restraining effect of the interval timer results in a gradual transition from parthenogenetic to gamic reproduction over a period of several generations and is still operational in the autumn. However, in this species even relatively long day conditions (17 h) can induce the development of oviparae. This low threshold of response to day-length combined with the short generation time results in the sexual morphs appearing very early in the year. This is of considerable adaptive significance in years when, as frequently happens, the aphids disappear locally before the onset of autumn.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Cytokine production in whole blood cell cultures of patients undergoing therapy with biological response modifiers or 5-fluorouracil

We have measured the levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-1, and IL-2 in the whole blood cell culture supernatants of 43 tumor patients undergoing a treatment with biological response modifiers or a conventional therapy with 5-fluorouracil and leucovorin. In the blood cell cultures of the 16 patients who received 5-fluorouracil and leucovorin IFN levels decreased (P0.01) and TNF levels rose (P0.05) during each therapy cycle. However, in the blood samples a declining number of total leukocytes and lymphocytes was measured (P0.05). Progressive disease could be correlated to a tendency towards lower IFN levels in the pretherapeutic cultures of these patients. The second group analyzed consisted of 8 patients receiving a low-dose IL-1 therapy. In this group we found either an unchanged or an augmented IFN production of the blood cells during treatment. In the group of 13 patients receiving low-dose recombinant IL-2 (4.5×10⁶IU m^–2 day^–1) significantly increasing IFN levels were seen in the blood cell cultures during the therapy (P0.05), although total leukocyte counts decreased. In this group, 4 had stable disease for at least 2 months and 9 patients had tumor progression under therapy. In the cultures of the latter a tendency towards lower IFN values was found. Finally, the cytokine production in the blood cell cultures of 6 patients receiving a combination therapy of IFN and high-dose IL-2 was studied. During this therapy a dramatically reduced production not only of IFN but also of all other measured cytokines was found. In this group all patients had tumor progression under therapy. It is concluded that the measurements of cytokine production in a reproducible whole blood culture system may be useful for monitoring immunological therapies and may help us to find out which doses of biological response modifiers have enhancing or suppressive effects on the functions of the immune cells.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Is ligation the only solution to the pyrophosphate problem?

Pyrophosphate linkages are easily formed during the nonenzymatic oligomerization of activated nucleotides. They often form caps which terminate an oligonucleotide with a 5-5 pyrophosphate. Owing to their structural resemblance to the intermediates in enzymatic ligation reactions, it has been suggested that pyrophosphate caps might have been capable of acting as activating groups in chain elongation processes. We argue that an alternative possibility would have been the specific hydrolysis of pyrophosphates.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Constant-time NOESY: An aid in the analysis of protein NMR spectra

Summary A constant-time version of the homonuclear NOESY experiment (CT-NOESY) is described. The experiment yields simplified protein spectra, in which cross peaks arising from protons with zero or small couplings are differentiated from other cross peaks, thus partially overcoming the problem of signal overlap. In addition, the CT-NOESY spectrum provides information on the magnitude of³J_NH- and³J coupling' constants, and is thus useful to determine torsion angle constraints and to perform stereospecific assignments of CHH protons in the case of³J constants.     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Triggering and predisposing factors in the “Red” decline syndrome of Norway spruce (Picea abies)

Summary Analysis of 62 mature Norway spruce (Picea abies provenance Viborg) trees growing in a Danish plantation was undertaken along with analysis of their nutrient contents (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B and Na), in each of the three youngest needle age classes, from branches of four exposure directions near the tree top. The aim was to investigate if one among the studied possible predisposing factors was also a triggering factor in the 1989 outbreak of the Red Norway spruce decline in Denmark. Neither nutrient imbalance or deficiency, nor excessive N-deposition or salt-stress were indicated as triggering factors in 1989. The Red syndrome, noticeable for the bright red colour of the current-year needles, was found to be an extension of the European type Novel Decline. Red syndrome is similar to previously reported phenomena of top-dying and sub top-dying, in that it had fewer needle age classes and significantly higher contents of mobile cations (and Ca) in the younger needle classes. Tree ring analysis suggested that the Red syndrome was initiated in the early 1980s, when the trees experienced adverse climatic conditions. Because of this long-term development of the Red Norway spruce decline syndrome, it is concluded that a triggering factor is of minor importance relative to the multitude of predisposing factors.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

The evolution of the type I interferon gene family in mammals

A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-, IFN-, and IFN-) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN- diverged first. Although apparent cases of interlocus recombination among mouse IFN- genes were identified, the hypothesis that coding regions of IFN- genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN- were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN- coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN- genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN- and IFN- is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN- genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Kinetic studies of peptide bond formation. Effect of chloramphenicol

There might be an undetermined order in the interaction of the substrates with the ribosomes in the reaction of CACCA-Leu-Ac with puromycin to form Ac-Leu-puromycin and CACCA (fragment reaction). Km 0°=6×10^-4 M for the puromycin · ribosome interaction. Chloramphenicol totally blocks the fragment reaction as a consequence of a single interaction with the ribosome of Kd 0°=2.2×10^-6 M. The inhibition by chloramphenicol of the fragment reaction is mixed competitive for puromycin.