Characteristics of alpha-aminoisobutyric acid transport in rat skeletal muscles.

alpha-Aminoisobutyric acid (AIB) transport into the intracellular compartment of extensor digitorum longus and soleus muscles was measured (in vitro) after allowance for the equilibration of the amino acid in the extracellular space. The latter was determined with three markers, [14C]inulin, 60Co-EDTA and [3H]mannitol. Net transport of AIB was subsequently divided into its two components, i.e. influx and efflux. Rates of influx were measured as the intracellular accumulation of [14C]AIB after a short incubation (5 min), and efflux was measured as the release of AIB with time (up to maximum of 50 min) from muscles that had previously been preloaded with AIB. This intracellular efflux was resolved into two phases, which probably represent two separate components of exit. The influence of extracellular Na+ on the transport of this neutral amino acid (representing the A system) was investigated. Na+ depletion resulted in lower accumulations of AIB, the effects becoming more pronounced with progressive depletions of external Na+. These changes arose from an inhibition of AIB influx, concomitant with an enhancement of its efflux. In contrast, all components of tyrosine transport (representing the L system) were unaffected by lowering external Na+ concentrations. The net accumulation of AIB was also suppressed by cortisol. This inhibitory effect was, however, Na+-dependent and resulted solely from the steroid's enhancement of AIB efflux, the hormone being without effect on AIB influx.     

Positive cooperativity in the adherence between elementary bodies of Chlamydia trachomatis strain UW-31 and HeLa cells

Abstract The adherence of purified elementary bodies of Chlamydia trachomatis strain UW-31 to monolayer cultures of HeLa 229 cells exhibited kinetic evidence of positive cooperativity. An abrupt increase in the rate of adherence occurred as chlamydial dose was increased. Only freshly isolated chlamydiae showed this behavior. In the presence of the lectin wheat germ agglutinin, the stimulated adherence showed typical Michaelis-Menten kinetics. The results suggest that chlamydiae may promote their own binding to the host-cell surface, and the lectin, when cell-bound, may provide additional chlamydiae-binding sites.     

σ Complexes as biochemical and biophysical probes: 4. Development of a novel reporter group delivery system

A novel reporter group delivery system for the chemical modification of proteins and the investigation of enzyme mechanisms is proposed. The design of this system is based on structural analogy with pyridoxal phosphate and σ-complex adduct formation. Progress is described toward the synthesis of suitable compounds, via three different approaches involving structural modification of the pyridine nucleus. A number of new compounds have been prepared, and other directions for future investigations are indicated.     

Duration of Hydrogen Formation by Anabaena cylindrica B629 in Atmospheres of Argon, Air, and Nitrogen

The time course of hydrogen formation by Anabaena cylindrica was followed beneath an argon atmosphere alone and also beneath atmospheres of argon, nitrogen, and air in the presence of carbon monoxide (0.2%) and acetylene (5%). Hydrogen production beneath argon alone was comparable in rate and duration (7 to 12 days) to that which occurred beneath air in the presence of carbon monoxide (0.2%) and acetylene (5%). However, much greater longevity (16 to 26 days) and improved rates of hydrogen formation were obtained when algae were incubated beneath argon and particularly nitrogen, each supplemented with carbon monoxide and acetylene. The total hydrogen produced by these cultures was up to three times as much as that released by cultures incubated beneath argon alone. Hydrogen-oxygen ratios for argon cultures either with or without carbon monoxide and acetylene were initially 1:5 but approximated 1:2 when measured over the entire incubation period. In each case oxygen production and nitrogenase activity (acetylene reduction) continued at reduced rates after hydrogen evolution had ceased. The effects of methionine sulfoximine (2 μM), ammonium ions (0.5 mM), or both on oxygen production were generally negligible, while effects on hydrogen production were variable depending on the atmosphere used; in most cases, eventual destabilization of the system occurred. A brief comparison was made of the time courses of anaerobic and aerobic hydrogen formation by the marine cyanobacterium Calothrix membranacea. It was found that shaking of cultures was beneficial for hydrogen production but not strictly necessary. It is concluded that hydrogen production by A. cylindrica in air and particularly nitrogen in the presence of carbon monoxide and acetylene offers the best potential of the atmospheres considered on the basis of four criteria: rates and longevity of hydrogen formation, practicality of the atmosphere used, and tolerance of hydrogen evolution to slight changes in composition of the atmosphere.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

2-carboxy-d-hexitol 1,6-bisphosphate: An inhibitor of d-ribulose 1,5-bisphosphate carboxylase/oxygenase

2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K_i at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the K_i was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO₂ for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K_i values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.