Genetics and ontogeny of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster

-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and hybrid forms of each, can be resolved. Analysis of F₂ and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.This project was supported in part by National Institutes of Health research grant GM-15597.     

Alpha-glycerophosphate dehydrogenase in Drosophila melanogaster: kinetic differences and developmental differentiation of the larval and adult isozymes

The isozymes of α-glycerophosphate dehydrogenase (α-GPDH) differ markedly with respect to their kinetic and stability parameters. Adult-limited GPDH-1 is stable at 50°C but decays at 57°C, while GPDH-3 is labile at 50°C under similar experimental conditions. By extrapolation of the thermal denaturation curves of crude adult extracts, we estimate GPDH-1 to constitute 76 per cent of the adult α-GPDH activity. Substrate kinetic studies revealed that, at pH 9·5, GPDH-3 exhibits an affinity for α-glycerophosphate which is twofold higher than that of GPDH-1, while K_m's for NAD⁺ are indistinguishable. The apparent K_m of GPDH-3 for dihydroxyacetone phosphate is consistently lower than that of GPDH-1 at pH 7·5, whereas at pH 6·7 the latter isozyme's apparent K_m approximates that of GPDH-3 at pH 7·5. Indistinguishable molecular weights of 66,000 were estimated by gel filtration for both GPDH-1 and 3. Gene dosage studies indicate that all three α-GPDH isozymes are simultaneously affected by dosage of the Gdh⁺ locus. These observations support a homomultimeric model of α-GPDH and the isozymes just discussed arise through epigenetic modification of the product of a single structural gene locus.     

A simple, one-step chromatographic procedure for the purification of lysozyme

A rapid method for the purification of lysozyme (mucopeptide N-acetyl-muramoylhydrolase, EC 3.2.1.17) from hen egg-white has been devised. It was that gel filtration chromatography on agarose columns can be used selectively to purify lysozyme, due to the fact that this protein interacts with the agarose matrix and elutes later than the corresponding total volume for the column. Thus, lysozyme is directly obtained in a relatively pure form and with a high specific activity. In principle, this simple method can be used to prepare lysozymes from other sources.     

Purification by dye-ligand chromatography and a crystallization study of the F1-ATPase and its major subunits, beta and alpha, from a thermophilic bacterium, PS3.

For a crystallization study, purification methods for F1-ATPase from a thermophilic bacterium, PS3, and its major subunits, beta and alpha, have been improved. The improvement depended on the introduction of dye-ligand chromatography columns to the previously adopted array of chromatography columns: a Blue-B (a blue dye bound to agarose) column was introduced for the F1 preparation, a Green-A column (a green dye attached to agarose) for the beta subunit, and a Blue-A (another blue dye, Cibacron Blue 3GA, bound to agarose) column for the alpha subunit. The improved preparations of all the proteins had purities of nearly 99%. Using the highly purified preparations of the proteins, crystallization conditions were searched for in a systematic way. Large plate crystals (0.2 X 0.5 X 0.5 mm) of F1 were grown from a polyethylene glycol solution. However, neither of the subunits was crystallized, in spite of extensive search for crystallization conditions.     

In vivo radiolabeling and turnover of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster: Gene dosage effects

In vivo radiolabeling of Drosophila melanogaster sn-glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8; GPDH) has been accomplished by microinjection of ³H-leucine into anesthetized flies. Comigration of immunoprecipitated radiolabeled GPDH with purified ¹⁴C-labeled GPDH-1 in SDS polyacrylamide disc gels has established the monospecificity of our immunoprecipitation technique. Short-term uptake experiments have demonstrated that maximum radiolabel incorporation of total TCA precipitable protein and immunoprecipitable GPDH-1 occurs within 4 hours postinjection, with GPDH-1 accounting for approximately 1% of the total radiolabeled TCA precipitable protein. In order to develop the parameters for turnover studies of GPDH in Drosophila, a comparative analysis of the rates of synthesis and degradation of GPDH-1 in flies bearing two and three doses of the structural gene have been conducted by the construction of adult flies aneuploid and euploid for the cytogenetic region 25F-26B on the left arm of chromosome II. Short-term uptake studies have demonstrated that the rate of GPDH-1 synthesis in the three-dose flies is approximately 1.58 times that found in the two-dose euploid flies. This value is in close agreement with data obtained for steady-state levels of CRM by rocket immunoelectrophoresis. In contrast, longterm pulse-chase experiments have revealed that rates of GPDH-1 degradation in these aneumploid and euploid flies appear to be identical. These data suggest that the rate of GPDH-1 synthesis in Drosophila is primarily regulated by a tightly linked cis-acting element which appears to act autonomously with respect to gene copy number as well as steady-state GPDH protein levels.     

Isolation of glycosylated hemoglobins by a combination of ion-exchange and gel-filtration chromatographies: a pilot study

The techniques of ion exchange and gel filtration have been combined in a single chromatographic column which allows the simultaneous isolation of hemoglobins glycosylated at their beta-amino termini from other hemoglobin species as well as from molecules differing in size from the hemoglobins. This method is unique because it makes possible isolation of preparative quantities of glycosylated hemoglobins within approximately 15 min. The method works most efficiently with a dry weight-to-weight ratio of Biorex 70 to Sephadex G25 of 1.4 to 1.0. The technique was applied to the determination of the apparent first-order rate constant for the deglycosylation of the labile form of hemoglobin AIc.     

A novel superporous agarose medium for high-speed protein chromatography

A novel superporous agarose (SA) bead characterized by the presence of wide pores has been fabricated by water-in-oil emulsification using solid granules of calcium carbonate as porogenic agent. After cross-linking, the solid granules were removed by dissolving them in hydrochloric acid. Then, the gel was modified with diethylaminoethyl groups to create an anion exchanger, SA-DEAE, for protein adsorption. A homogeneous agarose (HA) bead was also produced and modified with DEAE for comparison. It was found that the porosity of SA-DEAE was about 6% larger than that of HA-DEAE. Moreover, both optical micrographs and confocal laser scanning microscopy (CLSM) of the ion exchangers with adsorbed fluorescein isothiocyanate (FITC) labeled IgG revealed the superporous structure of the SA medium. In addition, the SA-DEAE column had lower backpressure than the HA-DEAE column, confirming the convective flow of mobile phase through the wide pores. Due to the presence of the wide pores, more channels were available for protein transport and, furthermore, more diffusive pores in the agarose network were accessible for the protein approach from different directions. This led to 40% higher protein capacity and two times higher effective pore diffusivity in the SA-DEAE than in HA-DEAE. Moreover, an increase of the efficiency of the SA-DEAE column until a flow rate of 5 cm/min and the independency of the column efficiency at flow rates from 5 to 17.8 cm/min was found, indicating that intraparticle mass transfer was intensified by convective flow at elevated flow rates. Therefore, the chromatographic resolution of IgG and BSA was little affected up to a flow rate of 17.8 cm/min. The results indicate that the SA medium is favorable for high-speed protein chromatography.     

Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

A simple procedure for regeneration of an organomercurial agarose column

Organomercurial agarose has been used in the purification of various thiol compounds including enzymes (1). Thiol compounds are first adsorbed on a column of organomercurial agarose, and then eluted with a second thiol compound, e.g., 2-mercaptoethanol (2-ME)¹ and cysteine. Although this column can be used repeatedly, a usual method for regeneration of the column is to remove the second thiol by HgCl₂. It would be desirable to regenerate the column without using HgCl₂, since it is biohazardous. In the study of the purification of a thiol-containing enzyme, we found that organomercurial agarose, which had previously been treated with 2-ME, could adsorb the enzyme and that the enzyme was eluted with 2-ME. This finding led us to examine whether the column can be used repeatedly without the regeneration using HgCl₂.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Immobilization of S-methyltransferase

S-methyltransferase was solubilized from pig liver microsomes by treatment with N-dodecyl-N,N-dimethyl-3-ammonio-1-sulfonate (Zwittergent). The soluble enzyme was immobilized by covalent binding to agarose and by copolymerization with acrylamide. The specific activity for the agarose-bound enzyme towards the substrate ethane thiol was 0.87 nmol/min/mg and for the acrylamide-bound enzyme 0.55 nmol/min/mg. The specific activity of the soluble enzyme was found to vary with increasing chain length of the substrate molecules from 0.5 nmol/min/mg for methane thiol (C1) to 6.3 nmol/min/mg for n-heptane thiol (C7). After binding of the enzyme to agarose beads, the increase in specific activity towards substrates with increasing chain length was no longer detectable. Instead, a relatively constant specific activity of 1.1 nmol/min/mg was observed for the whole range of substrates tested from C1 to C7. The stability of the agarose immobilized enzyme at -20 degrees C is twice as good as the soluble enzyme. The acrylamide immobilized enzyme is less stable than the soluble enzyme.     

Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products.

We have developed a procedure for the use of minislab gels to electrophoretically separate proteoglycans (PGs), large macromolecules with molecular masses greater than 2.5 million Da. Our procedure is a modification of the method of C.A. McDevitt and H. Muir (Anal. Biochem. 44, 612-622, 1971) for agarose/polyacrylamide, composite tube gels. These 1% agarose/1.2% acrylamide minigels are run at 35 mA for 75 min; bands are visualized by toluidine blue staining. The subtle size differences between the large aggregating PGs isolated from rat chondrosarcoma, bovine nasal septal cartilage, and adult bovine articular cartilage (which consists of two subpopulations) can be distinguished by their migration on these large pore gels. Chondroitin sulfate chains, added to all wells as a marker of constant mobility, ran immediately behind the dye front. The distance of migration into the gel of PGs incubated overnight with cathepsin B, carboxypeptidase A, papain, plasmin, elastase, or cathepsin G varied with the size of the cleavage products. We propose the use of this procedure for a convenient assessment of cartilage PGs and a rapid, reproducible assay for proteoglycanase activity.     

Three-dimensional spatial patterning of proteins in hydrogels

The ability to create three-dimensional biochemical environments that mimic those in vivo is valuable for the elucidation of fundamental biological phenomena and pathways. To this end, we designed a system in which proteins can be photochemically patterned in three dimensions within hydrogels under physiological conditions. Fibroblast growth factor-2 (FGF2) was immobilized within agarose hydrogels that were modified with two-photon labile 6-bromo-7-hydroxycoumarin-protected thiols. Two different methods were developed for FGF2 immobilization. The first procedure relies on the protein containing free cysteines for the formation of disulfide bonds with photoexposed agarose thiols. The second procedure takes advantage of the femtomolar binding partners, human serum albumin (HSA) and albumin binding domain (ABD), which have K(D) values of ~10(-14) M. Here HSA-maleimide was chemically bound to photoexposed agarose thiols, and then the FGF2-ABD fusion protein was added to form a stable complex with the immobilized HSA. The use of orthogonal, physical binding pairs allows protein immobilization under mild conditions and can be broadly applied to any protein expressed as an ABD fusion.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.