Willis A. Wood

Evidence for erythroid and nonerythroid forms of δ-aminolevulinate synthetase☆☆☆

Abstract The basis for the known transient accumulation of heme precursors during fetal development was investigated by comparing the characteristics of multiple forms of the first enzyme in the heme biosynthetic pathway, δ-aminolevulinate synthetase, derived from guinea pig fetal and adult liver and adult bone marrow. The elevation of δ-aminolevulinate synthetase in fetal liver was correlated with the transient establishment of erythropoietic activity in this organ during prenatal development. Partially purified mitochondrial synthetase from fetal liver and adult bone marrow (erythroid forms) was not bound to AMP- or CoA-agarose whereas the adult hepatic (nonerythroid) synthetase was strongly bound. For the first time, it was possible to demonstrate the identity of uninduced with 3,5-dicarbethoxy-1,4-dihydrocollidine-induced δ-aminolevulinate synthetase on the basis of the ability of both enzyme preparations to bind strongly to AMP-agarose. Weak binding of the erythroid forms to AMP-and CoA-agarose was observed in the presence of high concentrations of glycine. The erythroid forms had similar K0.5s for glycine (14 m m ) in contrast to the nonerythroid form, which exhibited a K0.5 of 3 m m and showed negative cooperativity. Additional kinetic studies suggested nucleotide regulation of enzyme activity. Sodium chloride (0.4 m ) strongly inhibited the erythroid enzymes but only slightly inhibited the nonerythroid synthetase. Furthermore, the specific activities of the crude extracts of the erythroid forms were 30 to 60 times that of the uninduced hepatic nonerythroid form. The apparent molecular weights of all three forms were approximately 110,000. The only significant difference between the two erythroid forms were their rates of heat inactivation at 45 °C.

A system for automatic recording of absorbancy and its application to enzyme-catalyzed reactions

Abstract A photometer of high stability and linear absorbancy characteristics is described. Attached to a conventional laboratory spectrophotometer like the Beckman DU, it permits direct recording of absorbancy changes with time. The device uses a photomultiplier in a special feedback system which permits flexible operation at full scale values from 0.1 to 3.0 or 4.0 absorbancy units. Linearity and reproducibility are of the order of 1% with a high order of base line stability. Detailed performance of the unit is described and typical velocity recordings of enzyme and chemical reactions are presented.

Distribution of C14 in sucrose from glycolate-C14 and serine-3-C14 metabolism☆

Abstract Serine-3-C 14 was converted by wheat leaves in the light to sucrose predominantly labeled in carbon atoms 1 and 6 of the hexoses. Glycolate-2-C 14 produced sucrose with C 14 hexoses labeled in carbons 1, 2, 5, and 6. By feeding glycolate-1-C 14 to soybean leaves, sucrose was obtained with 3,4-labeled hexoses. These results establish the existence of a glycolate pathway from the early carbon products of photosynthesis to uniformly labeled sucrose.

Synthesis of milk proteins in a cell-free system isolated from lactating bovine mammary tissue

Abstract Cell-free milk protein synthesis as measured by 14 C-leucine incorporation has been accomplished using partially defined components from lactating bovine mammary tissue. Incorporation of 14 C-leucine into trichloroacetic acid-precipitable protein and into specific milk proteins has been demonstrated. This synthesis is dependent on the presence of microsomes and an energy source and was partially dependent on the addition of sRNA and an aminoacyl-sRNA synthetase preparation. Incorporation was inhibited by ribonuclease, puromycin, cycloheximide, and sodium fluoride, but not by chloramphenicol and deoxyribonuclease. No stimulation is observed when various lactogenic hormones are added to the system. After addition of carrier, the isolated α-lactalbumin, β-lactoglobulm, κ-, α s -, and β-caseins were radioactive in about the same proportion as their abundance in milk and the isotope content remained relatively constant upon reisolation. Verification of α s -casein synthesis was obtained by a combination of radioautography and immunodiffusion procedures.

Recording of several simultaneous enzyme reactions facilitated by an automatic cuvette positioning attachment for the spectrophotometer

Abstract Two clock-operated devices for accurate sequential positioning of a cuvette carriage for the Beckman DU spectrophotometer is described. Through their use with associated equipment absorbancy changes resulting from reactions occurring in two to four cuvettes simultaneously can be recorded with a single-pen recorder. Data on reproducibility of positioning and typical recordings of enzyme-catalyzed reactions are provided.

Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase: IV. Structural features revealed by treatment with urea and Ellman's reagent☆

Abstract Native 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida and three other forms generated by experimental manipulation were investigated with respect to catalytic activity, reactivity with bisdithiodinitrobenzoate (Ellmans reagent), and, in some instances, were also partially characterized by optical rotatory dispersion measurements. These include: (a) the form present in 8 m urea; (b) the aldolase reconstituted after urea treatment, and (c) the ϵ- N -(1-carboxyethyl)-substituted aldolase generated by treatment with pyruvate and NaBH 4 . The native form had an apparent α-helix content of 31–33%. It reacted with 5.2 moles of Ellmans reagent per mole of aldolase without loss in activity. In the presence of 8 m urea, the α-helix content was zero and 12.1 moles of Ellmans reagent reacted per mole of aldolase. Thus, the four cysteinyl groups known to be present per subunit of the trimeric structure are distributed such that two are readily accessible and two are buried. The rate of reaction with Ellmans reagent depended upon the buffer present, as follows: HCO 3 > Tris = imidazole > arsenate ≧ phosphate > sulfate. Further, incomplete titration was observed in urea-phosphate as compared with urea-bicarbonate. After inactivation in 8 m urea-phosphate, as much as 90% of the original activity could be recovered. However, reactivation could not be achieved after treatment with urea-bicarbonate. The reconstituted aldolase behaved identically to the native aldolase by the criteria of titration with Ellmans reagent, density-gradient centrifugation, disc-gel electrophoresis, fractionation, crystal structure, α-helix content, and specific activity. Titration of ϵ- N -(1-carboxyethyl)lysyl 2-keto-3-deoxy-6-phosphogluconate aldolase with Ellmans reagent resulted in much lower reactivity of both free and buried SH groups. Analysis in urea density gradients revealed that dissociation in 8 m urea was very incomplete, thereby explaining the greater inaccessibility of thiol groups.

Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent-slope procedure: II. Application of the method to soluble and immobilized enzymes☆

Abstract A system is described for semiautomated evaluation of enzyme kinetic parameters, S0.5, V, and Hill n. It consists of a Gilford spectrophotometer modified for continuous addition of substrate to a stirred enzyme assay mixture, and for recording of absorbance data coded on paper tape. The Hill parameters for a number of enzymes, obtained from these data by tangent-slope or curve-fitting procedures, are in good agreement with published or manually determined values. Experiments with rabbit muscle lactate dehydrogenase covalently linked to Sephadex G-50 or Sepharose 4B demonstrate the feasibility of this approach when applied to enzymes attached to solid supports. Instrumental errors due to spectrophotometer nonlinearity and drift, stirring, mixing, pumping, truncation in readout of absorbance, and bias in experimental parameters are minimal and do not significantly interfere with the method. The advantages and disadvantages of the approach are discussed, and improvements and extensions are suggested for the instrumental and data handling system which could greatly extend its utility.

Determination of enzyme kinetic parameters by continuous addition of substrate to a single reaction mixture and analysis by a tangent-slope procedure: I. Analysis of the method using computed progress curves☆

A new method has been developed which provides reliable estimates of enzyme kinetic constants from single reaction progress curves recorded under conditions of continuously increasing substrate concentration. Equally spaced data points simulating such progress curves and containing known amounts of superimposed random noise were fit to the Hill equation by (i) direct nonlinear curve-fitting of raw data, and (ii) a tangent-slope technique in which the raw data are numerically differentiated, transformed into substrate versus velocity data, and then analyzed as linear plots. Both integral and differential procedures provided accurate and precise estimates of the Hill parameters (S0.5, V, and n) from single reaction mixtures. However, the tangent-slope method was at least 10-fold faster to compute and was not dependent on accurate initial guesses of the Hill parameters or integration of the rate equation. With the tangent-slope method, the optimal number of data points used in calculating tangent slopes was found to be 9 or 11. The reliability of the Hill parameters determined with the tangent-slope method was relatively insensitive to the maximum substrate concentration over a range of SmaxS0.5 of 1.5 to 10; the optimal value was 3. Through further analysis of simulated data, it was found that slow enzyme inactivation (<4% loss during the assay), or product competitive inhibition (maximum product concentration < 30% of the inhibitor dissociation constant) does not produce serious errors in the Hill parameters. Methods are presented to detect and distinguish enzyme inactivation and product competitive inhibition. It is suggested that continuous addition methodology combined with tangent-slope analysis provides the basis for a flexible system for kinetic characterization of enzymes which has wider applicability and other advantages over multicuvette or conventional progress curve methodology. A major advantage in contrast to the progress curve approach is that product accumulation and associated product effects are lowest at lower substrate concentrations.

An assay for δ-aminolevulinic acid synthetase based on a specific, semiautomatic determination of picomole quantitites of δ-[14C]aminolevulinate☆

Abstract δ-Aminolevulinic acid (ALA) synthetase activities in the range of 0.1 to 100 U/ml of enzyme are routinely assayed using a modified Beckman Model 121 amino acid analyzer. THis method reproducibly and specifically quantitates [14C]aminolevulinate from a mixture of [14C]succinate metabolites known to interfere in other methods. Mitochondrial ALA synthetase activity in livers of normal adult guinea pigs is determined to be approximately 3 U/g of liver. This activity is only 1 100 th the activity in guinea pigs treated with the porphyrinogenic drug 3,5-dicarbethoxy-1,4-dihydrocollidine.

Basic Biology of Microbial Fermentation

The great majority of what we know about microbial fermentations was discovered before 1950 (See review by Wood (1)). Since then microbial and biochemical science has passed on to questions of structure and function of macromolecules, organelles and cells thought to be more intriguing or deserving of attention. Now for economic and strategic reasons, attention is once again focused on microbial fermentations for their potential to produce useful and energy-containing products from starch, waste materials or currently unusable biomass. Further, strategists in this area have a kind of tacit belief that, somewhere in the tremendous explosion of biological and chemical knowledge since 1950, there are missing pieces of information and techniques, that when capitalized upon, will propel microbial fermentations from their current state of near obliyion back to center stage, being caught up in a new wave of bioinnovation. In such a scenario the task will be to couple the new knowledge with the old science and technology of fermentation. In such a new endeavor those with the old knowledge need to know what opportunities the new knowledge makes possible, and the practitioners of new science need to know a fair amount about the old science rather than attempting to “reinvent the wheel”.