Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

Production and characterization of aflatoxin B2a antiserum.

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B2a-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B2a-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B1 and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B12 and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B1 quantitation were 100 and 10 pg per assay, respectively.     

Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat

Previous studies in this laboratory showed that both alpha and beta receptors can mediate adrenergically-induced hyperglycemia in the cat. In the present study, the results of experiments on the isolated perfused cat liver provide affirmation that hepatic glycogenolysis in this species can be subserved by both types of receptors. Thus, the acute hepatic release of glucose induced by isoproterenol was found to be antagonized by propranolol but not by phentolamine or phenoxybenzamine. The opposite was found for the glycogenolytic action of phenylephrine. Experiments $\text{in}$$\text{vivo}$ showed that the hyperglycemic response to the beta agonist was associated with activation of hepatic phosphorylase and increased intracellular cAMP content while the hyperglycemia induced by the alpha agonist was associated with an activation of phosphorylase which was independent of cAMP.     

Cell cycle analysis and drug inhibition studies of silver staining in synchronous HeLa cells

Cytological staining with silver nitrate is specific for a protein associated with chromosomal nucleolus organizer regions and interphase nucleoli. At metaphase the amount of staining present is usually much less than that at interphase. During the transition from mitosis to G1, as seen in synchronized HeLa cells, the amount of silver staining increases and, by late G1, is located discretely and completely over the nucleolus. Such staining remains constant through G2. Towards late G2 a slight disorganization of the silver staining material is observed, possibly in preparation for the upcoming mitosis. Cells synchronized at mitosis and treated with either actinomycin D (AMD) or 2-mercapto-1-[2-(4-pyridyl)-ethyl]-benzimidazole (MPB), at concentrations which inhibit ribosomal RNA (rRNA) synthesis, show nucleolar fragmentation and little, if any, apparent increase in silver staining at early G1. After removal of the MPB, the nucleolar fragments reform nucleoli and the staining increases to control levels. Treatment of mitotic cells with puromycin dihydrochloride does not effect nucleolar morphology or the increase in silver staining. These results directly demonstrate that silver staining is associated with rRNA synthesis.     

Loss of tumorigenicity correlates with a reduction in pp60src kinase activity in a revertant subclone of avian sarcoma virus-infected field vole cells

We have recently isolated an interesting revertant subclone (revertant 866-4) of ESV-infected field vole cells that is indistinguishable from uninfected vole cells with respect to its lack of transformed cell properties. These revertants are not only normal morphologically, but they do not grow in soft agar and are nontumorigenic in athymic nude mice. Despite this lack of transformed cell properties, we have found that this cell line still contains pp60src at concentrations (0.30 microgram/mg cell protein) similar to those (0.13-0.42 microgram/mg cell protein) found in transformed and morphologically reverted, but tumorigenic vole cells (partial revertants). However, the most interesting aspect of this newly isolated subclone is the marked reduction in its pp60src kinase activity (2--3%) when compared with the specific activity of pp60src immunoprecipitated from transformed and partially revertant vole cell lines. Since the reduction in pp60src kinase activity strongly correlates with the loss of tumorigenicity in this particular revertant cell line, these data support the contention that this enzymatic activity is a crucial factor in the tumorigenic conversion of cells by avian sarcoma virus. Proteolytic peptide analysis of the structure of pp60src from revertant 866-4 indicates that it is similar to pp60src obtained from avian sarcoma virus-transformed chick embryo fibroblasts. Moreover, the reduction in kinase activity does not appear to be due to a lack of phosphorylation of the tyrosine residue in pp60src. Thus neither an obvious structural alteration nor a reduction in phosphorylation of pp60src appears responsible for the reduced kinase activity observed, suggesting that some as of yet undetermined feature of pp60src can influence the pp60src phosphorylating event.     

Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1.

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.     

Synergistic effect of kinetin on IAA-induced ethylene production

Kinetin has a significant synergistic effect on IAA-inducedehtylene production in hypocotyl sections of eitolated mungbean(Phaseolus mungo L.) seedlings when it is administered immediatelyafter cutting. If the addition of kinetin was delayed, it becameless effective. Kinetin-pretreated sections followed by incubationeither in IAA or in IAA plus kinetin produced ehtylene essentiallyat an identical rate to the one treated with IAA plus kinetinat zero time. However, the buffer-preincubated sections followedby incubation in IAA or in IAA plus kinetin shows a much reducedrate of ehtylene production as compared with the one treatedwith IAA or with IAA plus kinetin at zero time, respectively.These results suggest that kinetin is required for the maximalstimulation of ethylene production only during the early incubationperiod while IAA is required continuously throughout the incubation;the removal of IAA from the IAA-treated tissues causes an immediatecessation of ethylene production. ¹Present address: Mann Laboratory, Department of Vegetable Crops,University of California, Davis, California 95616, U.S.A. (Received May 10, 1973; )