Murine alpha-fetoprotein: N-terminal amino acid sequence and C-terminal residue.

We have purified to homogeneity murine alpha-fetoprotein (MAFP) and determined the amino acid sequence of the first twenty-four residues. The N-terminal sequence obtained shows a high degree of homology with human and rat AFP's, but not human or rat albumins. The C-terminal residue is the same as human and “slow” rat AFP, but different from the corresponding albumins. We conclude that the AFP's are derived from homologous genes which are at best distantly related to the ancestral gene for albumin. The single C-terminal residue and N-terminal sequence suggests that the multiple forms of MAFP observed by others are due to carbohydrate micro-heterogeneity.     

Picosecond dynamics of primary electron-transfer processes in bacterial photosynthesis.

The primary electron transfer processes in Rhodopseudomonas sphaeroides R-26 were studied as a function of temperature by means of picosecond spectroscopy. The first chemical step of the bacterial photosynthesis involves an electron transfer from the excited state of a bacteriochlorophyll a dimer, (BChl)2, to a bacteriopheophytin (BPh) to form the radical ion pair (BChl)2+. BPh-.. The upper limit for the formation time of this ion-pair was found to be 10 ps, at temperatures in the range 300-4.2 degree K. Similarly, the second chemical step, involving electron transfer from BPh-. to an ubiquinone-iron complex (QFe), was found to have a lifetime of approximately 150 ps, also independent of temperature in the same range. We interpret the absence of temperature dependence as indicating that process 2 proceeds via a tunneling mechanism. Utilizing our results in conjunction with electron tunneling theories, we calculate the distance between BPh-. and Q(Fe) to be 9--13 A. Our results also imply a closer proximity between (BChl)2 and BPh.     

Uptake and metabolism of tryptophan by the isolated perfused guinea-pig mammary gland.

Tryptophan uptake by the isolated perfused lactating guinea-pig mammary gland was 46.5+/-4.6 mug/h per g. Results of absorption studies and the use of [methylene-14C]tryptophan suggest that tryptophan is one of the group of amino acids that are transferred almost quantitatively from blood plasma to milk protein.     

The existence of a glyconeogenic pathway in rat skin.

The existence of a glyconeogenic pathway in rat skin has been demonstrated by measurement of three of the key glyconeogenic enzymes, fructose 1,6-bisphosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, and by studies on the incorporation in vitro of carbon from pyruvate and alanine into skin glycogen.     

Lectin-enzyme immunoassay of transferrin sialovariants using immobilized antitransferrin and enzyme-labeled galactose-binding lectin from Ricinus communis

A heterologous lectin-enzyme immunoassay is described. Microtiter plate wells were coated with affinity-purified antibodies to human transferrin. After incubation with transferrin sialovariants, prepared by limited neuraminidase treatment and separated with chromatofocusing, a lectin-enzyme-streptavidin complex was added. A good correlation was obtained between the number of terminal galactose groups on transferrin and the response in the lectin-enzyme immunoassay using Ricinus communis agglutinin as the galactose-binding lectin. The results indicate that characterization of glycosylation is possible with less than a microgram of the glycoprotein available, using lectin-enzyme immunoassays.     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.