The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.     

Monocyte-mediated augmentation of human natural killer cell activity: conditions, monocyte and effector cell characteristics

The characteristics of the effector cells and monocytes, and conditions required for the monocyte-mediated augmentation of human natural killer (NK) cell activity were investigated. Enriched null cell populations were further fractionated by Percoll centrifugation and used as effector cells. The LGL-enriched fraction was less susceptible than either the unfractionated cells or the other Percoll fractions to the monocyte augmentation when mixed with monocytes in the chromium-release assay and when precultured with monocytes for 12 hr, retrieved by carbonyl iron treatment, and tested for NK activity against K562. This differential susceptibility was reflected at the single cell level. The LGL-enriched Percoll fraction did not display the increase in target-binding cells with lytic activity that was exhibited by the other effector cell preparations after culture with monocytes. No differences in Leu-7 and Leu-11 phenotypes were detected between enriched null cells that had been cultured with and without monocytes for 12 hr. At the monocyte level, it was shown that pretreatment of the monocytes with LPS did not alter their NK-augmenting activity appreciably. Glutaraldehyde-fixed monocytes were not effective, and actinomycin D-treated monocytes were less effective than untreated or irradiated monocytes when mixed with enriched null cells in the assay. Actinomycin D-treated monocytes did not augment and possibly suppressed NK activity tested after 12-hr culture, and irradiated monocytes were less effective for augmenting NK activity than untreated cells. Monocyte-mediated augmentation could be detected when the medium used for null cell-monocyte coculture was supplemented with a) different lots of fetal bovine serum, b) human AB serum, c) autologous serum, or d) no serum. Polymyxin B and indomethacin did not alter the monocyte effect. Finally, the monocyte-mediated augmentation of human NK was not MHC restricted, since allogeneic combinations were also effective. These results suggest that 1) lymphocytes other than LGL participate in the monocyte-mediated augmentation of NK activity, 2) the augmentation is probably activational rather than maturational, 3) the monocytes must be viable to be effective when mixed with null cells during the assay, 4) de novo RNA and/or protein synthesis by the monocytes is required for the monocytes to induce augmented activity in null cells after 12-hr coculture, 5) prostaglandin synthesis and endotoxin are probably not involved in the augmentation, 6) the phenomenon is not MHC restricted, and 7) monocytes may express augmentative and suppressive activities concurrently.     

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

alpha-Bungarotoxin immobilized and oriented on a lipid bilayer vesicle surface

We have developed a new method to assess the binding site on alpha-bungarotoxin (alpha-BGT) for the acetylcholine receptor. It involves the covalent attachment of a palmitic acid chain to the toxin molecule, generating monopalmitoyl-alpha-bungarotoxin (PBGT) which is then immobilized on the surface of a lipid vesicle by a process of spontaneous insertion via the acyl chain into preformed unilamellar vesicles (approximately 800 A in diameter). PBGT itself is able to bind specifically to Triton X-100 solubilized acetylcholine receptors with an association constant, KA, of 5.56 X 10(6) M-1 which is approximately 20-fold lower in affinity than native alpha-BGT. Vesicle-associated PBGT binds to acetylcholine receptor enriched microsac membrane vesicles in aqueous buffer with a KA for both lipid and protein of 4.26 X 10(7) M-1. The putative site of acylation on the PBGT molecule is determined by extensive cleavage of a reduced and carboxymethylated PBGT with thermolysin. An acylated fragment is purified by hydrophobic column chromatography and identified by high-pressure liquid chromatography methods from the known primary sequence of the native toxin as a decapeptide including residues Thr47-Glu56 [C. Y. Lee convention used; see Mebs, D., Narita, K., Iwanaga, S., Samejuma, Y., & Lee, C. Y. (1971) Biochem. Biophys. Res. Commun. 44, 711-716]. Sequential hydrolysis of the fragment from the carboxy terminus with carboxypeptidase Y indicates that Lys51 is the sole site of acylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.