Biochemical Affinities Research Articles

Development of Biochemical and Cellular Probes to Study RIPK1 Target Engagement.

RIPK1 inhibitors have emerged as promising candidates for treating diverse diseases, including inflammatory diseases, autoimmune disorders, Alzheimer's disease, and cancer. However, the previously reported binding assays have limited sensitivity and stability, impeding high-throughput screening and robust characterization of the RIPK1 inhibitors. To address this challenge, we introduced two probes, T2-BDP-FL and T3-BDP-FL, derived from distinct RIPK1 inhibitors with different binding modes to establish time-resolved fluorescence resonance energy transfer (TR-FRET) displacement assays. Employing our TR-FRET displacement assays, we quantified the biochemical binding affinities of a series of RIPK1 inhibitors with diverse structural and binding modes for human RIPK1. Consistent results were obtained with these two probes in the TR-FRET displacement assay. Furthermore, we developed a RIPK1 fluorescent probe, T2-BDP589, for the NanoBRET assay. This assay enabled the characterization of RIPK1 target engagement by various RIPK1 inhibitors for both human and mouse RIPK1 in live cells. Our developed fluorescent probe displacement assays offer a sensitive and high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities.

Differential nuclear import sets the timing of protein access to the embryonic genome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins’ access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry.

Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (Kd = 1.7 μM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular Ki = 22 nM), docetaxel (cellular Ki = 16 nM), cabazitaxel (cellular Ki = 6 nM), and ixabepilone (cellular Ki = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (Kb) of the microtubule destabilizers colchicine (Kb = 80 nM, α = 0.08), vinblastine (Kb = 7 nM, α = 0.18), and maytansine (Kb = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (Kb = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.

Computational prediction of the long-term behavior of the femoral density after THR using the Silent Hip stem

Aseptic loosening due to the progressive periprosthetic bone resorption following total hip replacement is a crucial concern, that causes complications and failure of the arthroplasty surgery. The mismatch in stiffness between the hip implant and the surrounding femoral bone is one of the key factors leading to bone density resorption. This paper aimed to investigate the long-term response of the femoral bone after THR using the Silent Hip stem. For this purpose, a validated thermodynamic-based computational model was used to compute the change in bone density before and after THR. This model incorporated essential factors involved in bone remodeling process, such as mechanical loading, and biochemical affinities. The results of the numerical simulations using 3D finite element analysis were analyzed in five zones of interest qualitatively and quantitatively. Bone density predictions showed notable bone resorption in cervical areas, specifically in zone 1 and zone 5 of −18.7% and −14%, respectively. Conversely, bone formation was observed in the greater trochanter area (zone 2) of +25%. Stress shielding seemed to occur at cervical area due to the reduction in the mechanical loading in this region. Based on the quantitative analysis of the bone density distribution throughout the femoral bone, it appears that the Silent Hip stem achieved less bone resorption compared to conventional hip stem designs reported in the literature, which could be used for active patients.

SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein-ACE2 complex.

The newly reported Omicron variant is poised to replace Delta as the most prevalent SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, resulting in similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion. The increase in antibody evasion, together with retention of strong interactions at the ACE2 interface, thus represent important molecular features that likely contribute to the rapid spread of the Omicron variant.

Multiple pharmacological inhibitors targeting the epigenetic suppressor enhancer of zeste homolog 2 (Ezh2) accelerate osteoblast differentiation.

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.

Molecular complexity of visual mapping: a challenge for regenerating therapy.

Investigating the cellular and molecular mechanisms involved in the development of topographically ordered connections in the central nervous system constitutes an important issue in neurobiology because these connections are the base of the central nervous system normal function. The dominant model to study the development of topographic maps is the projection from the retinal ganglion cells to the optic tectum/colliculus. The expression pattern of Eph/ephrin system in opposing gradients both in the retina and the tectum, labels the local addresses on the target and gives specific sensitivities to growth cones according to their topographic origin in the retina. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. However, several lines of evidence have shown that the mapping can adjust to experimentally modified targets with flexibility, demonstrating the robustness of the guidance process. Here we discuss the complex ways the Ephs and ephrins interact allowing to understand how the retinotectal mapping is a precise but also a flexible process.

Long-term response of femoral density to hip implant and bone fracture plate: Computational study using a mechano-biochemical model

Although bone fracture plates can provide appropriate stability at the fracture site and lead to early patient mobilization, they significantly change the loading pattern in the bone after union (Stress shielding). This phenomenon results in a bone density decrease, which may cause premature failure of the implant. This paper presents the first study that quantifies the long-term response of femoral density to hip implantation and plating (lateral and anterior plating) using a mechano-biochemical model which considers the coupling effect between mechanical loading and biochemical affinities as stimuli for bone remodeling. The results showed that the regions directly beneath the plate experienced severe bone loss (i.e. up to ∼ –70%). However, some level of bone formation was observed in the vicinity of the most proximal and distal screw holes in both lateral and anterior plated femurs (i.e. up to ∼ +110%). The bone under the plate was divided into six zones. With respect to bone remodeling response, the findings revealed that anterior plating was not superior to lateral plating since the maximum and average bone losses among the zones in the anterior plated femur (i.e. –36% and –24%, respectively) were approximately the same as their corresponding values in the lateral plated femur (i.e. –38% and –24%, respectively).

Inhibition of CREB phosphorylation by conjugates of adenosine analogues and arginine-rich peptides, inhibitors of PKA catalytic subunit.

Bisubstrate inhibitors of protein kinases associate simultaneously with two substrate-binding sites of the kinase and thus potentially possess better inhibitory potency and selectivity than inhibitors binding to only the conserved ATP-site of the kinase. We have previously used conjugates of adenosine analogues and arginine-rich peptides (ARCs) to develop proteolytically stable cell plasma membrane-permeable bisubstrate inhibitors whose biochemical affinities towards several basophilic protein kinases of the AGC group are in the picomolar range. The potency of bisubstrate inhibitors to affect the phosphorylation of proteins in living cells has been described in a limited number of publications. In this study, the effect of ARCs on the protein kinase A (PKA)-catalysed cAMP response element-binding protein (CREB) phosphorylation pathway was studied in living mammalian cells. Our results demonstrate that at low micromolar extracellular concentration N-myristoylated ARCs are capable of reducing the activity of transcription factor CREB through inhibition of PKA.

Biochemical Assays of CaM and PiP2 Interactions with KCNQ Channel Domains

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are involved in controlling the gating of KCNQ ("M-type") K+ channels. It is well established that CaM has affinity for both the A and B helices of the KCNQ proximal C-terminus, and previous work indicates that CaM may be constitutively bound to the B helix. KCNQ channels are also suggested to contain multiple PIP2-binding sites, including a region of basic residues between the A and B helices (A-B linker) and a basic region at the C-terminal junction with the S6 transmembrane domain (S6Jx). Using a novel approach to understand the mechanisms controlling the modulation of KCNQ channels, we compared the biochemical affinities of PIP2 and CaM for these corresponding domains. Isothermal titration calorimetry (ITC) revealed a high affinity of Ca2+/CaM for a peptide corresponding to the KCNQ3 B helix (Kd ∼82 nM), whereas Ca2+/CaM bound the A helix with a much higher Kd (∼2 μM). The interactions between apo-CaM and the A or B helix peptide were not strong enough to obtain Kd values. Analytical ultracentrifugation results indicated that PIP2 directly bound to the S6Jx peptide, whereas its interaction with the A helix-B helix linker peptide is, as yet, indeterminate. These findings support the idea that CaM binds both the A and B helices of the KCNQ C-terminal domain in a Ca2+-dependent manner. Since the S6Jx domain is known to be close to the cell membrane by many structural studies of K+ channels, these data also suggest that only a very small conformational change in the C-termini of KCNQ channels is required to alter its binding to membrane PIP2. We will present more biochemical results that should help define the structural and molecular mechanism of PIP2 and CaM actions on KCNQ channels.

“Stainomics”: Identification of mitotracker labeled proteins in mammalian cells

Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.

Au Coating of Carbon Nanofiber-Tipped SPM Probes for Immobilization of Thiolated Biomolecules

An argon-ion-induced carbon nanofiber (CNF) is expected to be an ideal nanocarbon for developing probes for scanning probe microscopes (SPM) owing to its superior physical properties and structures. To use this pristine CNF as a biofunctionalized SPM probe, its surface needs to be further modified with probing molecules that have specific biochemical affinities toward biological targets. In this study, using a carbon-coated Si wafer as a substitute for a CNF-tipped SPM probe, we investigated ion sputtering for the formation of an Au film on a carbon surface, followed by thermal annealing for making the Au-crystal orientation preferable for the chemisorption of thiolated biomolecules. Finally, we confirmed the biofunctionality of the immobilized thiolated oligonucleotide probe on the annealed Au film in order to hybridize it with the complementary oligonucleotide target. The hybrids were detected by fluorescence emitted from streptavidin-conjugated quantum dots tagged to the biotin molecules. From these results, it was shown that Au coating, thermal annealing, and the subsequent immobilization of thiolated biomolecules can be used for the development of new biofunctional CNF-tipped SPM probes.

Nuclear Exportin Receptor CAS Regulates the NPI-1–Mediated Nuclear Import of HIV-1 Vpr

Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (VprN17C74) contained a bona fide nuclear localization signal and it is targeted VprN17C74 to the nuclear envelope and then imported into the nucleus by importin α (Impα) alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to VprN17C74. This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Impα) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1–mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.

Arabidopsis Profilin Isoforms, PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profilin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive localizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.

148 POSTER Comparison of the cellular and biochemical properties of ansamycin and non-ansamycin based Hsp90 inhibitors

Background: Heat shock protein 90 (Hsp90) has emerged as an important target for the treatment of cancer due to its essential role in several key oncogenic signaling pathways. Several classes of Hsp90 inhibitors have recently advanced into clinical trials including ansamycin derivatives that are semi-synthetic derivatives of the natural product geldanamycin (e.g. 17-AAG, IPI-504, 17-DMAG) or synthetic small molecules (e.g. purine derivatives, isoxazoles, pyrazoles). Ansamycin derivatives are potent Hsp90 inhibitors that demonstrate selective cell growth inhibition toward cancer cells as compared to normal cells. We have determined the biochemical and cellular properties of a group of published Hsp90 inhibitors, including both natural product derived and synthetic compounds. Materials and methods: The biochemical affinity of inhibitors to Hsp90 was determined using a competition binding assay using radioactively labeled competitor compound and Hsp90 purified from Hela cells. The growth inhibition induced by Hsp90 inhibitors was evaluated in human normal and cancer cells by measuring cell growth in the presence of varying concentrations of compounds using the Alamar Blue assay after 72h of compound addition. Results: The biochemical affinities to purified Hsp90 for the inhibitors tested range from 0.1 to 500 nM. There is an approximate correlation between biochemical affinity and cell growth inhibition of cancer cells with a relative activity ranking of isoxazoles > ansamycins > purines. While ansamycins demonstrate selective growth inhibition of cancer cells compared to normal cells as previously described in the literature, the more potent isoxazole derivative also potently inhibits the growth of some normal cell types. Three of the most potent compounds on cancer cells were also tested in vivo for their ability to induce the degradation of a Hsp90 client protein (mutEGFR) in a xenograft model of human NSCLC. In this model, we observe similar suppression of mutant EGFR and similar induction of cleaved caspase 3 for all three compounds, independent of their biochemical affinity for purified Hsp90. One possible explanation for this observation is provided by our measurements of off-rates of inhibitors from Hsp90. Conclusion: The experiments presented above raise the question of whether synthetic Hsp90 inhibitors with high affinity for Hsp90 have lost some of the in vitro therapeutic window between cancer and normal cells that makes Hsp90 inhibitors such attractive candidates for cancer therapeutics. Inhibitor Ki (nM)

Grand canonical Markov model: A stochastic theory for open nonequilibrium biochemical networks

In this paper we present the results of a stochastic model of reversible biochemical reaction networks that are being driven through an open boundary, such that the system is interacting with its surrounding environment with explicit material exchange. The stochastic model is based on the master equation approach and is intimately related to the grand canonical ensemble of statistical mechanics. We show that it is possible to analytically calculate the joint probability function of the random variables describing the number of molecules in each state of the system for general linear networks. Definitions of reaction chemical potentials and conductances follow from inherent properties of this model, providing a description of energy dissipation in the system. We are also able to suggest novel methods for experimentally determining reaction fluxes and biochemical affinities at nonequilibrium steady state as well as the overall network connectivity.

Biochemical affinities between Cucumis hystrix Chakr. and two cultivated Cucumis species (C. sativus L. and C. melo L.) based on isozyme analysis

Isozymes of eight enzymes in Cucumis hystrix Chakr. and two cultivated Cucumis species (C. sativus L. and C. melo L.) were analysed electrophoretically to investigate the biosystematics of these three species. Cluster analysis using data from six enzymes indicated that considerable genetic distance existed between both C. hystrix and melon and between C. hystrix and cucumber. C. hystrix might be a key species for studying the evolution and taxonomy of genus Cucumis.

Utility of Random Amplified Polymorphic DNAs for Determining Intraspecific Relationships in Cucumis

Principal component analyses of variation at 21 isozyme and 43 random amplified polymorphic DNA (RAPD) loci in eight cucumber (GY-14a, G421, H-19, WI 2757, and PIs 432860, 458845, and 183967) and seven melon [Top Mark (TM), Doublon, Green Flesh Honeydew (GFH), Juane Canari (JC), Freeman cucumber (FC), Snakemelon (SM), and PI 124111] cultigens were used to determine the use of these markers for assessing genetic variation among and within populations of each species (outgroup = Cucumis metuliferus). RAPD and isozyme marker variation was related to previous taxonomic classification and available pedigree information. Although dendrograms derived from cluster analyses using species' variation at marker loci were dissimilar, these disparities were consistent with differences in the pedigrees and/or other information (e.g., morphological) known about each accession and species. Elite U.S. processing cucumbers (G421, GY-14a, and H-19) shared distinctive biochemical affinities. Doublon was differentiated from TM, GFH, and JC. Doublon had biochemical affinities with FC, SM, and PI 124111.

Applications for Cryopreserved Blood Vessels in Pharmacological Research

The development of drugs in cardiovascular research involves in vitro studies that are generally performed on freshly isolated blood vessels. Naturally, data obtained from human tissues would be the most accurate and predictable for human pharmacology. However, the main problems are that the supply of fresh human material is unpredictable and the tissue has a very short life span. Storage in physiologic salt solution at 4°C, the most common practice to preserve isolated blood vessels, induces rapid and progressive changes of physiologic and functional properties within a few days. Cryopreservation of isolated blood vessels at - 70 to - 196°C in a medium containing Me 2SO offers the prospect of virtually infinite storage of the material with the only damage being that associated with the freezing and thawing process. Despite certain changes, such as reductions in contractile forces and endothelial functions, the main biochemical properties, uptake mechanisms, and affinities of most agonists and antagonists have been shown to be well preserved after cryopreservation. Hence, this technique offers clear potential for ensuring the supply of vascular material for pharmacological studies.

Phylogenetic relationships among several African Cucumis species

Cotyledons of seven African species within Cucumis, subgenus Melo, subgroup Myriocarpus (4x C. aculeatus, C. anguria var. anguria Meeuse, C. dipsaceus Ehrenb. ex Spach, C. ficifolius A. Rich, C. myriocarpus ssp. leptodermis Schweik., C. prophetarum, and 2x and 4x C. zeyheri Sond.), and 2x C. sativus L. of subgenus Cucumis were surveyed using horizontal starch gel protein electrophoresis to characterize interspecific and intraspecific variation. Cluster and classification and regression tree analysis of allelic frequencies among 14 polymorphic loci indicated that C. myriocarpus ssp. leptodermis, C. prophetarum ssp. dissectus, C. ficifolius, C. anguria var. anguria, and 2x and 4x C. zeyheri have biochemical affinities and could be distinguished from C. aculeatus and C. dipsaceus, which were similar. Within the first group, C. myriocarpus ssp. leptodermis and C. prophetarum ssp. dissectus accession formed a well-defined group. Cucumis prophetarum ssp. dissectus was more similar to C. myriocarpus ssp. leptodermis than to the C. ficifolius accessions evaluated. In the second group, two C. zeyheri accessions (C81321, C80346) were more similar to C. prophetarum ssp. prophetarum (C82069) than four other C. zeyheri evaluated. Cucumis sativus was dissimilar from all other species studied. Key words: evolution, biochemical genetics, isozymes.