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.     

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.     

Kinetics and specificity of alginate lyases: Part I, A case study

Purified preparations of alginate lyase from Klebsiella aerogenes and Haliotis sp. were investigated for activity and degradation patterns with alginate and alginate fragments having different compositions and sequences. With fragments approaching homopolymers of guluronate and mannuronate, Michaelis-Menten kinetics were obeyed and kinetic parameters could be obtained. Degradation of alginates containing all four possible linkages in various proportions, followed by isolation of the fragments and identification of the end groups by n.m.r. spectroscopy, indicated that the enzyme preparations can attack more than one type of linkage. The results are discussed with reference to the concept of specificity for enzymes with copolymeric substrates having non-regular distributions of units.     

Preparation and application of a photoreactive thrombin analogue: binding to human platelets

alpha-Thrombin has previously been shown to bind to specific, saturable glycoproteins on the platelet surface. Modification of the thrombin active site with tosyllysyl chloromethyl ketone (TosLysCH2Cl) does not alter thrombin's binding characteristics. Interaction of alpha-thrombin with high-affinity binding sites (KD = 10(-9) M) initiates the platelet response which involves proteolytic hydrolysis of this glycoprotein. Although TosLysCH2Cl--thrombin binds to and competes for the same sites as alpha-thrombin, it cannot induce platelet stimulation because it is enzymatically inactive. In this study, we describe the preparation and application of photoreactive tritium-labeled thrombin analogues. The alpha-thrombin derivative retains its platelet-stimulating and enzymatic activities and, upon photoactivation, covalently binds to specific platelet membrane components. When freshly washed human platelets are exposed to less than saturation doses (less than or equal to 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturating doses (greater than or equal to 20 nM) of the thrombin derivative yields a similar complex as well as two additional ones. Molecular weight estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4--polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl--thrombin has an approximate molecular weight of 200 000, while that with active alpha-thrombin is smaller, approximately 120 000, due to enzymatic cleavage. The additional complexes detected with the high thrombin dose had estimated molecular weights of 400 000 and 46 000, respectively, and appeared to be the same for TosLysCH2Cl--thrombin and for the alpha-thrombin coupled platelets. These isolated complexes appear to correspond to the two previously detected populations of thrombin binding sites on the platelet.     

Oocyte-follicle cell relationships in a starfish

Summary The follicle cells which surround the oocytes of starfish are known to both release the hormone 1-methyladenine and to respond to it by an active movement which forms a component of the spawning response to the hormone. In Patiria miniata these flagellated cells are located peripheral to the oocyte and have long cytoplasmic processes which penetrate the vitelline layer to the egg surface to form an adhering zonule-like junction. Within the follicle cell cytoplasm are located elongate filamentous bands which appear to represent a component of the contractile mechanism that mediates follicle cell response to 1-methyladenine. These bands do not resemble the filaments of vertebrate smooth muscle cells (quantity, distribution and size of filaments; lack of dense bodies in the filament mass), nor the contractile units of the superficial epithelium of lower vertebrate follicles.This investigation was supported by grants HD-07194 and HD-12499 from the National Institutes of Health. We are indebted to Mr. James D. Huber for able technical assistance     

DNA FLOW CYTOMETRY OF ISOLATED KERATINIZED EPITHELIA: A METHODOLOGICAL STUDY BASED ON ULTRASONIC TISSUE DISAGGREGATION

Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G₁, S and G₂+ M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions. This method of preparation prior to DNA flow cytometry was useful for the study of the hamster cheek pouch epithelium and of normal and pathological human epidermis.     

Plasma membrane Ca2+ transport: stimulation by soluble proteins.

Inside-out membrane vesicles were prepared from human red blood cells. In the presence of ATP, these vesicles took up ⁴⁵Ca²⁺ against a chemical gradient. The active transport of Ca²⁺ was increased by addition of an activator protein of (Ca²⁺+Mg²⁺)-ATPase isolated from the membrane-free hemolysate of human red blood cells. A closely related protein, the protein modulator of cyclic AMP phosphodiesterase from bovine brain, also increased the rate of active transport of ⁴⁵Ca²⁺. Addition of the calcium ionophore A23187 caused a rapid efflux of ⁴⁵Ca²⁺ from loaded, inside-out vesicles. When La³⁺ was added to the system in the presence of activator protein, the uptake of ⁴⁵Ca²⁺ was inhibited. Results are compatible with the interpretation that activity of the plasma membrane Ca²⁺ pump may be modulated by certain cytoplasmic proteins.     

Ultrastructural evidence of maintenance of concanavalin A binding sites in enucleated mammalian cells

Mouse L cells were enucleated by centrifugation in cytochalasin B. Following enucleation, the enucleated cells were incubated in fresh medium for 30 min, 4, 20, or 24 h before being fixed for electron microscopy. After fixation the cells were incubated in concanavalin A and then horseradish peroxidase was bound to the ConA. Electron microscopy of these enucleates revealed that the concanavalin A-binding sites (CABS) are present on the cell surface of the enucleates even at 24 h after enucleation. Although the method does not detail the number of sites present, the inherent distribution of sites appears similar in normal and enucleated cells. Furthermore, the sites are still functional in that the live enucleated cells are agglutinated by ConA to the same extent as are normal L cells—about 80% agglutination in each instance. The results of this study indicate that surface CABS are maintained in the absence of a nucleus and they are still present even after the Golgi apparatus is morphologically disrupted. Turnover of these sites and their relationship to nuclear function are discussed.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.