Harvey Kaplan

Purification and characterization of a trypsin-like digestive enzyme from spruce budworm (Chroristoneura fumiferana) responsible for the activation of δ-endotoxin from Bacillus thuringiensis

A trypsin-like enzyme purified from spruce budworm (Choristoneura fumiferana) gut juice has a molecular mass of 25 kDa and its pH activity profile indicates a pKa of 8. Sequence homology with bovine trypsin of the N-terminus and active site, and the ionization dependence for catalysis, reflect the typical trypsin-like activities measured. The action of this enzyme (designated CFT-1) is compared to the neat gut juice with regard to the proteolytic activation of the delta-endotoxin from Bacillus thuringiensis.

Covalent cross-linking of proteins without chemical reagents

A facile method for the formation of zero‐length covalent cross‐links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross‐linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85°C for 24 h. Under these conditions, approximately one‐third of the total protein present becomes cross‐linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero‐length cross‐links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross‐linked dimer has only one amide cross‐link and retains the enzymatic activity of the monomer. The in vacuo cross‐linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross‐linking, and co‐lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross‐linked dimer.

Activation of teratocarcinoma-derived hemoglobin genes in teratocarcinoma-friend cell hybrids

Hybrid cells formed by the fusion of murine teratocarcinoma and Friend erythroleukemia cells synthesize hemoglobin in the presence of chemical inducers such as dimethylsulfoxide (DMSO). By making use of the fact that the parental teratocarcinoma and Friend cells carried different alleles at the locus coding for the alpha chain of hemoglobin, it was possible to demonstrate that the teratocarcinoma-derived genes for the globin alpha chains are genetically active in hemoglobin-synthesizing hybrid cells. In addition, evidence is presented suggesting that the teratocarcinoma-derived genes for the beta-globin chains may also be expressed in the hybrids. Apparently the teratocarcinoma-derived genome has become reprogrammed to express erythroid functions following fusion of the teratocarcinoma cell to the Friend cell.

Role of DNA in the Activation of the Cry1A Insecticidal Crystal Protein from Bacillus thuringiensis

The Cry1A insecticidal crystal protein (protoxin) from six subspecies of Bacillus thuringiensis as well as the Cry1Aa, Cry1Ab, and Cry1Ac proteins cloned in Escherichia coli was found to contain 20-kilobase pair DNA. Only the N-terminal toxic moiety of the protoxin was found to interact with the DNA. Analysis of the crystal gave approximately 3 base pairs of DNA per molecule of protoxin, indicating that only a small region of the N-terminal toxic moiety interacts with the DNA. It is proposed that the DNA-protoxin complex is virus-like in structure with a central DNA core surrounded by protein interacting with the DNA with the peripheral ends of the C-terminal region extending outward. It is shown that this structure accounts for the unusual proteolysis observed in the generation of toxin in which it appears that peptides are removed by obligatory sequential cleavages starting from the C terminus of the protoxin. Activation of the protoxin by spruce budworm (Choristoneura fumiferana) gut juice is shown to proceed through intermediates consisting of protein-DNA complexes. Larval trypsin initially converts the 20-kilobase pair DNA-protoxin complex to a 20-kilobase pair DNA-toxin complex, which is subsequently converted to a 100-base pair DNA-toxin complex by a gut nuclease and ultimately to the DNA-free toxin.

A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of δ-endotoxin from Bacillus thuringiensis subsp, sotto

A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the delta-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDS-PAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated delta-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of delta-endotoxin may play a role in the expression of activity towards various insect larvae.

Chemical Modification of Lyophilized Proteins in Nonaqueous Environments

Lyophilized proteins were reacted in vacuo with a volatile reagent or dispersed in octane and reacted with dissolved reagent. Three novel derivatives were formed with iodomethane: (a) quaternized trimethyl amino groups, (b) N1,N3-dimethylimidazolium cation, and (c) phenolic O-methyl ether. Acid anhydrides acylated amino groups and formed mixed anhydrides with side-chain carboxyl groups. Under nonaqueous conditions it was observed that: (i) The same derivatives are formed as under aqueous conditions. (ii) Hydrolytic breakdown of protein is prevented. (iii) Less reagent is required. (iv) Unreacted reagent can be recovered. (v) Water-labile derivatives can be isolated as stable intermediates. (vi) The yield of a derivatized functional group was directly related to its pKa, its surface exposure, and the pH of the solution from which the protein was lyophilized. (vii) The physicochemical factors governing the reactivity of protein functional groups in nonaqueous environments appear to reflect the protein solution structure prior to lyophilization.

Reactive properties of the amino groups of histones in calf thymus chromatin

Abstract The reactivity of the five main histones in calf thymus chromatin towards acetic anhydride has been determined using the technique of competitive labelling (Kaplan et al ., 1971; Kaplan, 1972). Under trace labelling conditions all the histones incorporate radioactive label from tritiated acetic anhydride, however, only histone IIb2 incorporates the label at a rate which is consistent with a substantial portion of its amino groups being fully exposed. Identification of the reactive functional groups of histone IIb2, revealed that the reactivity could be accounted for entirely by the reactivity of its N-terminal proline. It is concluded that the vast majority if not all the ϵ-amino groups of histones in calf thymus chromatin have abnormally high pK a values and thus appear to be buried, while the N-terminus of histone IIb2 is exposed. This conclusion supports the hypothesis that the ϵ-amino groups of histones form salt linkages with the phosphates of DNA.

Glycation Improves the Thermostability of Trypsin and Chymotrypsin

A novel glycation procedure, in vacuo glycation, was used to attach glucose covalently to the lysine residues of trypsin and chymotrypsin. Glycated trypsin and glycated chymotrypsin have greatly increased thermostability compared to the native enzymes. For example, glycated bovine trypsin, incubated at 50°C and pH 8.0 for 3 h, retained more than 50% of its original activity whereas the native enzyme was inactivated under the same conditions. Similarly, after incubation at 50°C and pH 8.0, glycated bovine chymotrypsin retained 45% of its original activity and the native enzyme was inactivated. Glycated porcine trypsin is exceptionally thermostable and could be used to digest native ribonuclease at 70°C without the need for prior denaturation. The apparent increase in the thermal stability of the glycated proteins observed in activity measurements is also reflected by an increase in the Tm values determined with differential scanning calorimetry (DSC) and circular dichroism (CD). The glycation does not alter the activity or specificity of these enzymes. Biotechnol. Bioeng. 2008;101: 452–459.

Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction.

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.

The toxic moiety of the Bacillus thuringiensis protoxin undergoes a conformational change upon activation

Proteolytic processing of the 133-kDa crystal protein (protoxin) from Bacillus thuringiensis subsp. kurstaki yields a 67-kDa insecticidal toxin. Differential scanning calorimetry was used to investigate whether the toxic moiety in the protoxin molecule has the same conformation as activated toxin. Compared to protoxin, toxin gives rise to a more complex endotherm which extends over a 10 degrees C broader temperature range and contains a component occurring at a substantially higher temperature than any unfolding transition in the protoxin endotherm. It is concluded that the toxic moiety undergoes a conformational change upon activation in which the thermal stability of at least one of its domains is significantly increased.