Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

Hydrogen evolution and uptake by nodules of soybeans inoculated with different strains of Rhizobium japonicum

Hydrogen evolved by nitrogenase may be recycled by a hydrogenase present in some legume nodules. Anoka and Portage cultivars of soybeans were inoculated with each of 8 and 24 strains, respectively, of Rhizobium japonicum and surveyed for H2 evolution and C2H2 reduction rates nodule weight, and plant dry weight. Six of the strains (3Ilb 110, USDA 122, USDA 136, 3Ilb 6, 3Ilb 142, and 3Ilb 143) which exhibited no H2 evolution in air were shown to take up H2. The relative efficiencies of nitrogenase energy utilization based on C2H2 reduction rates of nodules relative efficiences of nitrogenase energy utilization based on C2H2 reduction rates of nodules ranged from 0.96 to 1.0 for the six strains. Nodules formed by strain WA 5099-1-1 evolved small amounts of H2 in air and had a relative efficiency of 0.92. Nodules formed by the remaining 25 strains had relative efficiencies ranging from 0.41 to 0.80. A H2-evolving (3Ilb 123) and non-H2-evolving (3Ilb 143) strain were tested on seven soybean cultivars to determine the effect on the expression of hydrogenase. Nodules formed by strain 3Ilb 143 exhibited an efficiency of 1.0 on the following cultivars: Amsoy 71, Anoka, Bonus, Clark 63, Kent, Peking, and Portage. Relative efficiencies from 0.63 to 0.77 were determined for the five cultivars nodulated by strain 3Ilb 123. From the experiments with these cultivars, the capacity to recycle H2 produced from the nitrogenase system appears to be determined by the R. japonicum strain.     

Affinity chromatography of phenylalanine hydroxylase. The structure of a pteridine adsorbent

1. Four independent methods have established that the structure of a previously reported pteridine affinity adsorbent, 6,7-dimethyl-5,6,7,8-tetrahydropterin--CH-Sepharose, is 5(CH-Sepharosyl)-6,7-dimethyl-5,6,7,8-tetrahydropterin. 2. A novel reaction, the carbodiimide-promoted coupling of a carboxyl group to N-5 of a tetrahydropterin, is described. 3. Two novel adsorbents, 5-formyl-tetrahydrofolate--AH-Sepharose and 5-methyl-tetrahydrofolate--AH-Sepharose, are described which may be useful not only in the study of phenylalanine hydroxylase but also in the study of folate-metabolizing enzymes.     

Immune reactivity of frozen-thawed murine spleen cells.

Spleen cells from mice immunized with SRBC were subjected to controlled rate freezing to ?100 °C. Complete recovery of PFC was obtained with DMSO used as the cryopreservative. Simple dilution of spleen cells in DMSO, or a single cycle of freezing and thawing in DMSO prior to short-term culture, resulted in early loss of recoverable cells. A single cycle of freezing and thawing inhibited the in vitro immune response to SRBC while having little effect on the response to TNP-T4. The in vitro blastogenic responses to LPS and PHA-P were severely reduced in cultures of frozen and thawed cells.     

Reduction of experimental myocardial infarct size with hyperosmolar mannitol.

Hypertonic mannitol previously has been shown to improve cardiac function, increase collateral flow, and decrease epicardial ST segment elevation following coronary occlusion in anesthetized or awake dogs. The present study quantitates by morphologic techniques, the effect of hypertonic mannitol on infarct size. Ischemic injury was produced by proximal occlusion of the circumflex artery for 40 min and necrosis was assessed after 48 hr of reflow. One group of dogs was given isotonic saline and the other hypertonic mannitol beginning the infusions just prior to, during, and for a short period after the release of the circumflex coronary artery occlusion. Serum osmolality increased by approximately 40 mOsm in the mannitol group. The administration of hypertonic mannitol was associated with a 40-50% reduction in infarct size ventricular fibrillation during occlusion and following release of the circumflex coronary artery occlusion was greater in mannitol-treated dogs although the difference was not statistically significant. Thus, the data obtained in this study extend previous observations and provide direct evidence that hypertonic mannitol can reduce infarct size in dogs with temporary circumflex artery occlusion and reflow.     

The circular dichroic spectra of several sialic acid-containing polysaccharides isolated from neisseria meningitidis

The circular dichroic spectra of the acid and sodium salt forms of several sialic acid-containing homo- and hetero-polysaccharides have been measured. The spectra are shown to be influenced by the state of ionization of the carboxyl groups contained in the sialic acid, the location within the individual sialic acid residues of the inter-saccharide linkages, and changes in the configuration of hydroxyl groups remote to the carboxyl group of the sialic acid.     

Interaction between phloretin and the red blood cell membrane

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.     

Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance.

The application of 13-C nuclear magnetic resonance to the analysis of some sialic acid-containing meningococcal polysaccharide antigens is described. Complete assignments of the spectra of both the native serogroup B and the de-O-acetylated serogroup C polysaccharides have been made. These assignments were based on the corresponding data for some related monomers (sialic acid and its alpha-and beta-methylglycosides) and on supportive chemical evidence. The data indicate that the serogroup B polysaccharide is a 2 yields 8-alpha-linked homopolymer of sialic acid, identical in structure with colominic acid from Escherichia coli, whereas the de-O-acetylated serogroup C polysaccharide is a 2 yield 9-alpha-linked homopolymer. The native serogroup C polysaccharide is O-acetylated (1.16 mol of O-acetyl per sialic acid residue), all the O-acetyl substituents being located only at C-7 and C-8 of the sialic acid residues, and in addition contains unacetylated residues (24%). The polysaccharide contains di-O-acetylated residues (O-acetyl on C-7 and C-8), and at least one of the possible monoacetylated residues at C-7 or C-8.