A self-stabilized model of the chymotrypsin catalytic pocket. The energy profile of the overall catalytic cycle

A model of the catalytic triad of chymotrypsin is built assuring the arrangement and properties as they are within the complete enzyme. The model contains 18 amino acid residues of chymotrypsin and its substrate. A total of 135 atoms (including 70 heavy atoms) were subjected to full ab initio geometry optimizations through 127 individual steps along the reaction coordinate of the complete catalytic mechanism. It was shown that the described model of the catalytic apparatus forms a self-stabilized molecule ensemble without the rest of the enzyme and substrate. According to the calculations, the formations of the first and second tetrahedral intermediates in the model have 20.3 and 15.7 kcal/mol activation energy barriers, respectively. Removing elements of the catalytic apparatus such as the (1) catalytic aspartate or (2) the anion hole, as well as (3) inserting a water molecule "early" in the catalytic process, or (4) introducing conformational rigidity of the substrate, results in an increase of the above energy barrier of the first catalytic step in the model by 6.4, 13.7, 3.7, and 4.1 kcal/mol, respectively. Based on the calculated process one can conclude that the catalytic reaction in this model is much more similar to the reaction in the enzyme than to the reference reaction. To our knowledge, this is the first model system that mimics the complete catalytic mechanism.     

A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.     

Velocity of Ellman's reaction and its implication for kinetic studies in the millisecond time range

A detailed study of the velocity of the reaction between Ellman's reagent and thiocholine was undertaken, in order to test the possibilities of this reaction as a detection method for the earlier stages of cholinesterases reactions. Experiments were carried out on a stopped-flow apparatus with a built-in spectrophotometer. The obtained experimental data were analyzed by fitting the data to theoretical kinetic equations derived for the reaction. In this way, a complete kinetic characterization of the reaction was obtained. An important practical result derived from our investigations is the finding that, under most experimental conditions, the Ellman's reactions is more than sufficiently rapid as a detection method. However, in the case of reactions in the time scale of 200 milliseconds or less, this being 5 times the half life of Ellman's reaction at standard conditions, one has to consider the interference of this reaction with the enzyme reaction itself.     

Escherichia coli alkaline phosphatase. An analysis of transient kinetics

1. The hydrolysis of 2,4-dinitrophenyl phosphate by Escherichia coli alkaline phosphatase at pH5.5 was studied by the stopped-flow technique. The rate of production of 2,4-dinitrophenol was measured both in reactions with substrate in excess of enzyme and in single turnovers with excess of enzyme over substrate. It was found that the step that determined the rate of the transient phase of this reaction was an isomerization of the enzyme occurring before substrate binding. 2. No difference was observed between the reaction after mixing a pre-equilibrium mixture of alkaline phosphatase and inorganic phosphate, with 2,4-dinitrophenyl phosphate at pH5.5 in the stopped-flow apparatus, and the control reaction in which inorganic phosphate was pre-equilibrated with the substrate. Since dephosphorylation is the rate-limiting step of the complete turnover at pH5.5, this observation suggests that alkaline phosphatase can bind two different ligands simultaneously, one at each of the active sites on the dimeric enzyme, even though only one site is catalytically active at any given time. 3. Kinetic methods are outlined for the distinction between two pathways of substrate binding, which include an isomerization either of the free enzyme or of the enzyme-substrate complex.     

Theory for the design of apparatus for drying frozen tissues

A general theory for the design of apparatus for the low temperature drying of frozen tissues has been developed. Using this theory it is possible to compute drying rates for different materials in different apparatuses and to design an apparatus suitable for the required purpose. It is shown that for the drying of animal tissues in the laboratory, where the area is small, the requirements of the apparatus are not particularly critical. The same theory applied to the drying of biologicals and plasma indicates that the design is exceedingly critical and suggests directions in which existing apparatus might be modified. Aided by a grant from the Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago and from the Commonwealth Fund of New York.     

Effects of gamma-irradiation on the cardiovascular system of sheep

In experiments with sheep, the response of the vascular system of the mammals to the total exposure dose was studied. A dose increase resulted in a more pronounced impairment of contraction of the cardiac muscle. In response, a compensatory reaction of the peripheral vessels was intensified up to LD50/30. Higher doses led to a complete disorder in the regulatory apparatus of the circulatory system.     

New technique for studying reaction forces during primate behaviors on vertical substrates

Recording reaction forces from primates during behaviors on vertical substrates, such as leaping, climbing, or biting trees, typically requires the design and construction of customized recording devices or mounting commercially available force platforms in a vertical position. The technical difficulties imposed by either option have hindered in vivo research on the kinetics of primate behaviors on vertical substrates. We describe a simple, inexpensive apparatus for recording forces from primate behaviors on vertical substrates. The apparatus includes an instrumented beam fastened directly to a horizontal force platform and a surrounding vertical substrate that does not contact the instrumented beam or platform. The contact piece at the end of the instrumented beam is positioned flush with the noninstrumented vertical substrate, and reaction forces elicited on this instrumented section are directed to the force platform. Because most of the vertical substrate is not instrumented, we can isolate and record forces from a single limb or jaw during a behavior. Biewener and Full ([1992] Biomechanics Structures and Positions: A Practical Approach; New York: Oxford University press, p. 45-73) gave seven criteria to consider when designing a customized force-recording device. Where appropriate, we tested if our apparatus met their criteria. The apparatus accurately records forces in three orthogonal directions, has low cross-talk, maintains a high frequency response, exhibits a linear response up to at least 200 Newtons, and displays a uniform response to a given force across the instrumented contact piece. Our design does not easily facilitate the identification of the point of force application. Therefore, joint moments cannot be easily calculated. This limitation, however, does not affect the apparatus's ability to accurately record the magnitude and direction of a force (as shown by other tests). We developed this apparatus to measure jaw forces during tree gouging in common marmosets (Callithrix jacchus), but the general design can be readily modified to study a variety of primate behaviors on vertical substrates.     

A simple apparatus for the continuous monitoring of 14CO2 production from several small reaction mixtures

A simple apparatus is described which permits the continuous monitoring of ¹⁴CO₂ production from ten separate reaction mixtures simultaneously. The device is relatively simple and inexpensive to construct, makes use of small disposable incubation vials, and allows complete trapping of all ¹⁴CO₂ evolved in scintillation vials, where it can be easily counted. The use of this apparatus to determine the rates of metabolism by glomeruli of ¹⁴C-labeled substrates to ¹⁴CO₂ is described.     

A gas-flow cryostat for use in freeze-quench studies: design and application to discontinuous pre-steady-state spectral analyses

A design for an improved freeze-quench apparatus is presented. A freeze-quench apparatus has two main parts: the apparatus which mixes the reactants and after a set time sprays the reacting mixture into the quencher, and the quenching apparatus itself. The quenching apparatus is the novel feature of our design and it comprises a gas-flow cryostat mounted directly onto a liquid nitrogen storage Dewar. The gas flow maintains the temperature of a small volume of isopentane, which acts as the freeze-quench agent, at -140 degrees C. (The apparatus can maintain the quenching temperature over the range -190 degrees C to room temperature.) Because of the small size of the cryostat and the much reduced volume of solvent used by this method it is more convenient to use than its predecessors, can be used in the open laboratory, and is safer. Our apparatus is designed for application to electron paramagnetic resonance studies but could be easily modified for use with other spectroscopic techniques.     

Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation.

The role of the SEC7 gene product in yeast intercompartmental protein transport was examined. A spectrum of N-linked oligosaccharide structures, ranging from core to nearly complete outer chain carbohydrate, was observed on glycoproteins accumulated in secretion-defective sec7 mutant cells. Terminal alpha 1-3-linked outer chain mannose residues failed to be added to N-linked glycoproteins in sec7 cells at the restrictive temperature. These results suggest that outer chain glycosyl modifications do not occur within a single compartment. Additional evidence consistent with subdivision of the yeast Golgi apparatus came from a cell-free glycoprotein transport reaction in which wild-type membranes sustained outer chain carbohydrate growth up to, but not including, addition of alpha 1-3 mannose residues. Golgi apparatus compartments may specialize in addition of distinct outer chain determinants. The SEC7 gene product was suggested to regulate protein transport between and from functional compartments of the yeast Golgi apparatus.     

Kinetic bacteriochlorophyll fluorometer

A pump and probe fluorometer with a laser diode as single light source has been constructed for measurement of fast induction and relaxation of the fluorescence yield in intact cells, chromatophores and isolated reaction centers of photosynthetic bacteria. The time resolution of the fluorometer is limited by the repetition time of the probing flashes to 20 μs. The apparatus offers high sensitivity, excellent performance and can become a versatile device for a range of demanding applications. Some of them are demonstrated here including fast and easy investigation of the (1) organization and redox state of the photosynthetic apparatus of the intact cells of different bacterial strains and mutants and (2) electron transfer reactions on donor and acceptor sides of isolated reaction centers. The compact design of the mechanics, optics, electronics, and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems.     

Observations Relating to the Efficiency of Mixing in Rapid Reaction Flow Devices

The principle of dimensional analysis of liquid flow has been applied to the problem of rapid mixing in flow apparatus. A model of the Hartridge-Roughton mixing chamber and observation tube has been scaled approximately 1/1.000 so that the times after mixing are approximately 6 s, the flow velocities are the order of 2 cm/s, and the distance from mixing to observation is 120 mm. Visual observation is employed to observe the end point of the mixing of acid-base with a color indicator, thymol blue. Quantitative estimates of the effect of the number of jets, the effect of screens placed in the flow stream, and the angle of jets, one with respect to another, lead to quantitative evaluations of these parameters for the scale model. The extent to which these parameters apply to the full-scale apparatus at faster flow velocities suggest that the general principles employed in the scale model are valid, although more emphasis is placed upon turbulence generation at the low flow velocities employed in this experiment than at the faster flow velocities employed in the full-scale apparatus.     

Morphological and videofluorographic study of the hyoid apparatus and its function in the rabbit (Oryctolagus cuniculus)

The anatomy of the hyoid apparatus and positional changes of the hyoid bone during mastication and deglutition are described in the New Zealand White rabbit (Oryctolagus cuniculus). A testable model is constructed to predict the range of movement during function of the hyoid, a bone entirely suspended by soft tissue. Frame-by-frame analysis of a videofluorographic tape confirms the accuracy of the prediction through observation of hyoid bone excursion during oral behavior. During chewing, translation of the hyoid bone is diminutive and irregular, lacking a clearly discernible path of excursion. However, some movements of the hyoid occur with regularity. During fast opening, anterodorsal movement of the hyoid is interrupted with an abrupt posteroventral depression when the bolus is moved posteriorly toward the cheek teeth by the tongue. This clockwise rotation (when viewed from the right side) of the hyoid accompanies jaw opening and is reversed (posteroventral movement) for the jaw closing sequence. Lateral movements of the hyoid may be slightly coupled to mandibular rotation in the horizontal plane. The findings suggest that the hyoid bone maintains a relatively static position during the dynamics of chewing. The primary function would be to provide a stable base for the movements of the tongue. Another possible function would be to control the position of the larynx within the pharyngeal cavity. Some characteristic features of the rabbit hyoid apparatus may be consequential to relatively erect posture and a saltatory mode of locomotion.     

ULTRASTRUCTURAL LOCALIZATION OF CALCITONIN IN THE PARAFOLLICULAR CELLS OF PIG THYROID GLAND WITH CYTOCHROME c-LABELED ANTIBODY FRAGMENTS

Parafollicular cells in mammalian thyroid glands are thought to be responsible for the secretion of calcitonin. In this study, calcitonin was localized in pig thyroid gland by an indirect immunocytochemical technique using rabbit antiserum directed against synthetic porcine calcitonin for the first step, and sheep Fab fragments prepared against rabbit Fab and coupled to cytochrome c for the second step. The antigenic determinants of calcitonin were present only in the parafollicular cells, whose secretory granules were heavily labeled. Labeling of the cytoplasmic matrix is thought to indicate a possible leakage of the polypeptide from the granules. A striking observation was the complete absence of labeling in the cisternae of the rough-surfaced endoplasmic reticulum and of the Golgi apparatus. It is concluded that the secretory granules of parafollicular cells contain calcitonin; the mechanism of synthesis of this peptide is not clearly understood.     

An apparatus for continuous analysis of protease-catalyzed acyl transfer reactions

An apparatus that allows continuous analysis of protease-catalyzed acyl transfer reactions is described. Hydrolysis reaction is assayed using automatic titration. A continuous determination of amino group concentration by reaction with o-phthalaldehyde gives the rate of peptide bond formation. The apparatus allows the determination of the partition constant for the nucleophile at various nucleophile concentrations from one run.     

Measurement of the Water Potential of Intact Plant Tissues1

A modified design of a thermocouple psychrometer is describedwhich utilizes the Peltier effect for producing an exceedinglysmall wet-bulb thermometer. The thermocouple assembly has beenreduced to miniature dimensions and the thermocouple chamberconsists of a silver cylinder (volume 0.06 cm³) whose base isformed by the material under observation. This makes it possibleto measure water potentials over limited surfaces of intactplant tissues, e.g. germinating pea seeds (Pisum sativum L.).The apparatus is capable of measuring water potentials downto a magnitude of about –6,000 joules/kg.³     

Genetic and physiological analysis of induced late mutants ofarabidopsis thaliana (l.) heynh

Using four independent late mutants, obtained by treatment of the early cultivar Dijon-G with N-methyl-N-nitrosourea, it was found that their delayed flowering has a monogenic genetic basis, the mutant alleles appearing to be incompletely dominant. All these mutants display a positive reaction to the graded vernalisation; the vernalisation requirement of the individual mutants is, however, both quantitatively and qualitatively different and hence specific for each of the mutant genotypes. lodoacetic acid is found to inhibit the vernalisation of the mutants to some extent only when the duration of the vernalisation is relatively short (20 days). A hypothesis is advanced, assuming a potential existence of a complete genetic apparatus for vernalisation already in the Dijon-G cultivar, where it is, however, blocked by suppressor or epistatic genes.     

A generalization of kinetic data on sulphite oxidation systems

Kinetic data of the reaction between sulphite ions and dissolved oxygen depend on the purity of sulphite used. Thus it is adequate to determine them for each batch and concentration of sulphite used. Besides the dependence of reaction rate constants on catalyst concentration, it is usually also required to know their dependence on pH and on temperature. Both quantities can change to a great extent especially in absorption apparatus in which high sulphite conversion occurs. They profoundly influence the value of the reaction rate constant. To get a complete picture of the influence of these three quantities without repeating extensive kinetic measurements for every kind and concentration of sulphite used, a method for obtaining information on the influence of all three quoted quantities in the range of values of interest for application is presented.     

Cloning and characterization of beta-COP from Dictyostelium discoideum

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.     

High-pressure stopped-flow fluorometry at subzero temperatures: application to kinetics of the binding of NADH to liver alcohol dehydrogenase

A stopped-flow apparatus operating in fluorescence mode over temperature and pressure ranges of +30 to -30 degrees C and 10(-3) to 2 kbar, respectively, is described. The system was interfaced on a special spectrofluorometer. Its general design is an improvement of the previous instrument (C. Balny, J. L. Saldana, and N. Dahan, (1984) Anal. Biochem. 139, 178-189) in that the observation chamber and the driving mechanism have been modified. The application of the method to kinetics of the binding of NADH to horse liver alcohol dehydrogenase at subzero temperatures and as a function of hydrostatic pressure is described.