Ferenc J. Kezdy

Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta

Abstract A new sensitive method for determining juvenile hormone (JH) hydrolysis has been developed which measures the release of tritiated methanol from JH labelled in the methyl ester group. Using this assay we investigated the interaction of JH with haemolymph esterases and haemolymph JH-binding protein. Haemolymph from fifth instar larvae of Manduca sexta contains two families of esterases which can be distinguished by their reactivity with diisopropylphosphorofluoridate (DFP). One group consists of general esterases which are capable of hydrolysing free JH but not JH complexed to the binding protein and are completely inhibited by low concentrations of DFP (10 −4 M). The other group (JH-specific esterases), relatively DFP resistant, has little detectable general esterase activity but can hydrolyse JH bound to the binding protein as well as free JH. The major JH-esterase has a sedimentation coefficient of 4·98 S and a diffusion coefficient of 6·4 × 10 −7 cm 2 sec −1 . The molecular weight calculated from these values is 6·7 × 10 4 . The general esterases are present throughout the larval stage, but the JH-specific esterases are barely detectable until the fourth day of the fifth instar when they suddenly appear at a high concentration. Since the general esterases cannot hydrolyse bound JH, one function of the binding protein is to protect JH during transport in the early instars, thus confirming that the binding protein is a true carrier of JH. In the late fifth instar prior to metamorphosis, however, JH-specific esterases appear in the haemolymph resulting in the hydrolysis of JH complexed to the carrier protein. Thus, by lowering JH titre, the JH-esterases play an important role in development in M. sexta .

Isolation and characterization of a larval lipoprotein from the hemolymph of Manduca sexta.

1. The larval high density lipoprotein (HDL) from the hemolymph of Manduca sexta, isolated by density gradient centrifugation, contains 61% protein, 37% lipid and 2% carbohydrate. 2. The molecular weight of HDL is 6 x 10(5), with two apoproteins of 2.85 x 10(5) and 8.1 x 10(4) daltons. 3. The large apoprotein is destroyed by trypsin treatment of the particle, while the small one is not. 4. Calculations based upon size and composition show that this particle is very different in structure from mammalian lipoproteins. It is proposed that a portion of the apoprotein occupies the central core region.

Chromogenic substrates and assay of phospholipases A2

Publisher Summary This chapter focuses on the chromogenic substrates and assay of phospholipase A 2 . The high specificity of phospholipases A 2 (PLA 2 ) toward aggregated substrates renders the assay of phospholipase A 2 a particularly challenging analytical task. Sensitive assays require the use of aggregated substrates, such as micelles, mixed micelles, single bilayer vesicles, or monomolecular layers, where the activity of the enzyme depends critically on the physical state and the exact composition of the nonaqueous phase. For reproducible assays with such heterogeneous systems, the experimental conditions must be strictly controlled as the presence of minor lipid impurities, the accumulation of small amounts of reaction products in the early phases of the reaction, or even slight changes in temperature or buffer composition may and do elicit large changes in the rate of the enzymatic reaction. Homogeneous reaction kinetics, which are conducive to readily reproducible kinetics, could only be achieved with short-chain lecithins, such as dibutyryllecithin, toward which phospholipases A 2 display a rather low specificity, and the assays based on these substrates are not sensitive enough for most purposes. The chapter describes a spectrophotometric assay, which lies between the two extremes, and which by virtue of its simplicity, is readily adaptable to a variety of purposes ranging from routine analyses to investigations of mechanistic details of the enzymatic reaction. This assay provides a rapid, accurate, and convenient method for measuring phospholipase A 2 concentrations as low as 1 × 10 -8 M.

Polymer-encased vesicles derived from dioctadecyldimethylammonium methacrylate

Dispersal of dioctadecyldimethylammonium methacrylate (DODAM) in water via ultrasonic irradiation yielded small-diameter vesicles having a phase transition at ca. 42-46/sup 0/C. Photopolymerization (254 nm) at 30 and 60/sup 0/C resulted in the formation of polymer-encased vesicles which retained phase-transition behavior. Combination of dynamic light scattering, electron microscopy, and captured volume data provides strong evidence for vesicle shrinkage when polymerization is carried out at 60/sup 0/C; shrinkage occurring during photopolymerization at 30/sup 0/C is less certain. Poly(methacrylic acid), derived from 30/sup 0/-polymerized vesicles (30/sup 0/-polymerized means polymerized at 30/sup 0/C in this paper), was 75.4% syndiotactic, 22.3% heterotactic, and 2.3% isotactic and was significantly more soluble in DMF than poly(methacrylic acid) derived from 60/sup 0/-polymerized vesicles; the latter polymer was predominantly syndiotactic. At 25/sup 0/C, nonpolymerized, 30/sup 0/-polymerized, and 60/sup 0/-polymerized vesicles showed similar permeability toward sucrose; at 60/sub 0/C, the 60/sup 0/-polymerized vesicles were less permeable. Storage of the 60/sup 0/-polymerized and the nonpolymerized DODAM vesicles for 2 months at room temperature revealed the former to be more stable. The monolayer properties and photopolymerization behavior of DODAM have been investigated at the air-water interface.

Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis

The activity of 3-hydroxy-3-methylglutaryl CoA reductase in homogenates of the corpora allata of the tobacco hornworm, Manduca sexta, was competitively inhibited by compactin. The KI for the sodium salt form of compactin was 0.9 nM for the reductase from both male and female corpora allata. In intact female corpora allata juvenile hormone biosynthesis was also inhibited by approximately 50 percent at 10 nM compactin. Following injection with compactin, darkening of the cuticle, an indication of juvenile hormone deficiency, was observed in larvae after ecdysis from third to fourth instar. Hence, compactin shows potential as an inhibitor of insect growth and development.

Biochemical Aspects of Juvenile Hormone Action in Insects

Insects undergo profound morphological changes that take place long after the embryonic stage and are thus excellent models for the study of extrinsic control of genetic programs in higher organisms. In the process of metamorphosis, undifferentiated cells organized as imaginal disks develop into complex structures (legs, wings, antennae, etc.). The genetic program that dictates the ecdysone-induced differentiation and development of the imaginal discs is regulated and modulated by the juvenile hormone (JH). A high titer of juvenile hormone prevents differentiation and rapid growth of the disks. Prior to metamorphosis the hormone titer falls, and the low level then permits development to proceed. Thus, juvenile hormone exerts an inhibitory effect on the course of development during larval life.

A method for probing the affinity of peptides for amphiphilic surfaces

We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.

Characterization of carotene accumulation inUstilago violacea using high-performance liquid chromatography

Quantitative analysis of carotene accumulation in white, pink, pumpkin, orange, and yellow haploid strains ofUstilago violacea by high-performance liquid chromatography indicated that specific patterns of carotene accumulation are primarily responsible for the white, pumpkin, orange, and yellow phenotypes. The yellow strains accumulated primarily β-zeacarotene and β-carotene. The white strains accumulated primarily the colorless carotene, phytoene, or did not accumulate any carotene at all. Carotene accumulation in pink haploid strains followed the same patterns as for the white, pumpkin, orange, or yellow strains. Pink diploid and disomic strains ofU. violacea with various parental combinations of the color mutations accumulated either cis-β-zeacarotene and β-carotene or only β-carotene. The pattern of carotene accumulation in conjunction with the available genetic information for the carotene loci inU. violacea was used as a basis for the construction of a new genetic model for carotene biosynthesis inU. violacea. The model employs three dehydrogenases and one cyclase for the synthesis of β-carotene from phytoene, and accounts for the carotene accumulation patterns of either cis-β-zeacarotene and β-carotene or lycopene, γ-carotene, and β-carotene.

Characterization of the pineapple stem proteases (bromelains).

The two major acidic sulfhydryl proteases, bromelain A and bromelain B, from the pineapple stem have been characterized by their kinetic properties toward p-nitrophenyl esters of N-α-acyl amino acids. Both enzymes show the same broad specificity toward the amino acid side chains at pH 4.7. Less than an order of magnitude of difference is observed in the rates of bromelain A-catalyzed hydrolyses of the following five amino acid esters: Z-l-lysine (kcat = 7.4 s−1, Km = 57 μM), Z-l-alanine (kcat = 2.5 s−1, Km = 24 μm), Z-l-tyrosine (kcat = 0.4 s−1, Km = 7.6 μm), Z-glycine (kcat = 1.75 s−1, Km = 174μm) and Z-l-asparagine (kcat = 1.5 s−1, Km = 75 μm). Valine, leucine, isoleucine, and tryptophan derivatives react at least one-thousand times slower. The pineapple stem acetone powder also contains sulfhydryl proteases of markedly different specificity. A purification procedure of bromelain A and B is described which eliminates these enzymatic impurities.

The reaction of tris (hydroxymethyl) aminomethane with calf intestinal alkaline phosphatase

The effect of tris (hydroxymethyl) aminomethane concentrations on the rate of calf intestinal alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate was studied, in the pH range 8-10, where no transphosphorylation reaction could be detected. Kinetic analysis of the results permitted description of the effect of Tris concentrations T on the rate of enzyme catalyzed hydrolysis (V) by the following equation: (see article). The rate-accelerating effect of Tris concentrations can be ascribed to two different mechanisms: At moderate Tris concentrations (0.01-0.20 M) the enzyme forms a reversible addition complex with a Tris molecule. This complex has an enhanced catalytic activity. We suggest that the binding of Tris to the enzyme could potentiate a second active site of the enzyme, due to its ionization effect upon an acidic group of the enzyme of pK = 8.9. The modest linear rate accelerating effect of Tris at high concentrations (0.20-0.60 M) could be ascribed to the change of the dielectric constant of the medium, the degree of solvation of the protein, or change in the tertiary structure of the enzyme.