The synthesis of radioactive 3-methylthiopropionate and other alkylthio fatty acids.

Aprocedure is described for the synthesis of radioactive 3-methylthiopropionate, a recently isolated metabolite of mammalian methionine metabolism. The method is a two-step synthesis whereby correspondingly labeled methionine is degraded by ninhydrin to 3-methylthiopropionaldehyde and then specifically oxidized to 3-methylthiopropionate without oxidation of the sulfur atom by the yeast enzyme, aldehyde dehydrogenase. Radiochemical purity of the isolated product was established by paper, thin-layer, and gas-liquid chromatography. This procedure is economical and readily applicable to the synthesis of other alkylthio fatty acids for the study of S-methylcysteine and ethionine metabolism and probably for the synthesis of radioactive intermediates of branched chain amino acids.     

The electrolytic preparation of bioactive radioiodinated parathyroid hormone of high specific activity.

A technique for the preparation of microgram quantities of bovine parathyroid hormone (bPTH) labeled with carrier-free ¹²⁵I to a specific activity of 1300 Ci/mmol is described. A restructured and simplified apparatus was used for electrolytic iodination, making it feasible to use reaction volumes of 100 to 200 ml. The miniaturized setup requires only a small platinum crucible connected via an agar-KCl salt bridge to a saturated KCl solution, a battery to drive the reaction, and a voltmeter to monitor the potential difference between the reference-saturated KCl solution (via a calomel electrode) and the platinum crucible. The [¹²⁵I]-labeled bPTH elutes as a single species when chromatographed on a Biogel P-10 column equilibrated in 3 m guanidine HCl-2.3 m formic acid, and it retains full biologic activity when bioassayed in vivo. It is evident that bPTH labeled to a high specific activity with ¹²⁵I does not suffer in regard to its biological potency.     

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions.

An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic E_max is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H₂SO₄ is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Purification of the creatine kinase isozymes of the green sunfish (Lepomis cyanellus) with Blue Sepharose CL-6B.

Three homodimeric creatine kinase isozymes (A₂, B₂, and C₂) of the green sunfish (Lepomis cyanellus) were purified by a combination of affinity chromatography, gel filtration, and preparative starch gel electrophoresis. The final preparations were isozymically pure and were used to elicit antibodies in rabbits. The use of the group-specific adsorbant Blue Sepharose CL-6B (Pharmacia) and specific elution conditions for creatine kinase facilitated purification. Fish creatine kinase isozymes are sensitive to denaturation and cannot be readily purified by procedures routinely used for mammalian creatine kinase isozymes.     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

A rapid and specific fluorescent activity staining procedure for transamidating enzymes.

A rapid, sensitive, and specific procedure has been developed for detection of transsamidatingenzymes (transglutaminase, e.g., factor XIII) after agarose gel electrophoresis. The technique is based on the transamidase-catalyzed incorporation of the fluorescent monodansylthiacadaverine into casein. The high sensitivity enables detection and characterization of transamidases in blood plasma, platelet, and red blood cell lysate and tissue extracts. The technique can also be combined with crossed immunoelectrophoresis.     

The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membranes.

The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.