Gradients of Intercellular CO(2) Levels Across the Leaf Mesophyll

Most current photosynthesis models, and interpretations of many wholeleaf CO₂ gas exchange measurements, are based on the often unstated assumption that the partial pressure of CO₂ is nearly uniform throughout the airspaces of the leaf mesophyll. Here we present measurements of CO₂ gradients across amphistomatous leaves allowed to assimilate CO₂ through only one surface, thus simulating hypostomatous leaves. We studied five species: Eucalyptus pauciflora Sieb. ex Spreng., Brassica chinensis L., Gossypium hirsutum L., Phaseolus vulgaris L., and Spinacia oleracea L. For Eucalyptus, maximum CO₂ pressure differences across the leaf mesophyll were 73 and 160 microbar when the pressures outside the lower leaf surface were 310 and 590 microbar, respectively. Using an approximate theoretical calculation, we infer that if the CO₂ had been supplied equally at both surfaces then the respective mean intercellular CO₂ pressures would have been roughly 12 and 27 microbar less than the pressures in the substomatal cavities in these cases. For ambient CO₂ pressures near 320 microbar, the average and minimum pressure differences across the mesophyll were 45 and 13 microbar. The corresponding mean intercellular CO₂ pressures would then be roughly 8 and 2 microbar less than those in the substomatal cavities. Pressure differences were generally smaller for the four agricultural species than for Eucalyptus, but they were nevertheless larger than previously reported values.     

Movement of Fe with respect to the heme plane in the R-T transition of carp hemoglobin. An extended x-ray absorption fine structure study

Carp Hb undergoes a well known change in kinetics over the pH range 6-9. X-ray absorption spectroscopy, in conjunction with refined data analysis procedures, shows no difference in iron-ligand distances when carp HbCO is switched from R (high affinity) to T (low affinity) states. These distances are 2.015 +/- 0.015 A for the average iron-pyrrole nitrogen distance, 2.14 +/- 0.04 A for the iron-nitrogen (of histidine) distance, and 1.89 +/- 0.05 A for the Fe-C (of CO) distance. Examination of the region from 30 to 100 eV above the threshold, called the ligand field indicator region, reveals spectral differences, which when compared to model compounds suggest that the iron and the heme are less coplanar in the T-like forms. These results are consistent with the iron being 0.1 A more out of the mean heme plane in both carp HbCO and carp Hb T states, relative to the R forms, and that the change in iron position on ligation to either T or R state is four times larger than that occurring with the quaternary switch.     

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., L?nngren, J., Arnarp, J., Haraldsson, M., & L?nn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand binding kinetic studies on the hybrid hemoglobin alpha (human):beta (carp): a hemoglobin with mixed conformations and sequential conformational changes

Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) alpha (human):beta (carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human alpha-chain is R state and the carp beta-chain is T state. We have no evidence at this pH that the carp beta-chain ever assumes the R conformation. At pH 6, the human alpha-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp beta-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human alpha-chains are in the R state, the two beta-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the beta-chains first assume T-state properties, followed by the alpha-chains. The rate of the R-T conformational change for the carp beta-chains is at least 300 times greater than that for the human alpha-chains. At pH 9, the R----T conformational transition rate is at least 200 times slower than that for human hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.     

Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin

A small stopped-flow cuvette was built into a computer-controlled Cary 210 spectrophotometer. The enzymatic depletion of oxygen in solutions of hemoglobin and myoglobin was initiated by flowing the hemeproteins with the enzyme against a solution of the hemeproteins containing the appropriate substrate. The deoxygenation was homogeneous throughout the solution. Oxygen activity was calculated at each instant of time from the fractional saturation of Mb, determined from observations at the Hb/HbO2 isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO2 isosbestic wavelength. The spectrophotometer cycled between these two wavelengths during the deoxygenation. The deoxygenation of HbO2 was largely complete in 20-25 min, whereas the deoxygenation of MbO2 was allowed to proceed for about 1 h. This procedure eliminates equilibration of Hb solutions with a gas phase and replaces oxygen electrode readings with spectrophotometric sensing by Mb, providing essentially instantaneous determinations of oxygen activity and hence 250-500 or more independent data points per run. The Mb and Hb data vectors require several manipulations to correct for small relative displacements in time and for small non-isosbestic effects. Detailed consideration of the enzyme kinetics allowed oxygen activities to be determined in regions where Mb is a poor sensor. Studies of HbO2 deoxygenation as a function of wavelength show that the determination of the four Adair constants requires in addition the determination of three spectroscopic parameters. Values of the apparent Adair constants, determined without these spectroscopic parameters, depend strongly on the monitoring wavelength.     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.