Distribution of Thylakoid Proteins between Stromal and Granal Lamellae in Spirodela : Dual Location of Photosystem II Components

We have quantified the lateral distribution of 12 thylakoid proteins of Spirodela oligorrhiza by immunoblot analysis of detergent-derived granal and stromal lamellae. The immunological, ultrastructural, cytochemical, and biophysical measurements each indicated the expected overall separation of photosystem II (PSII) and photosystem I (PSI) components; however, certain proteins were not completely localized to one lamellar fraction. The apoproteins of the light harvesting chlorophyll a/b complex, subunit 1 of PSI and the components of the PSII reaction center (the 32 kilodalton, D2, and cytochrome b₅₅₉ proteins) were dually located between granal and stromal lamellae. Proteins associated exclusively with one of the membrane types were: in granal lamellae, the 43 and 51 kilodalton PSII proteins, and in stromal lamellae, the α and β subunits of the proton ATPase.     

l-Glutamined-Fructose-6-P aminotransferase regulation by glucose-6-P and UDP-N-Acetylglucosamine

l-Glutamined-Fructose-6-P aminotransferase regulates hexosamine synthesis. An affinity purified human fibroblast aminotransferase and specific radioisotope assays developed by us were used to show an independent inhibition of the aminotransferase by Glucose-6-P. More interestingly, at concentration of UDP-N-Acetylglucosamine and glucose-6-P where either sugar has no independent inhibitory effect, there is an allosteric and significant inhibition of the aminotransferase.     

Oxygen quenching of sensitized terbium luminescence in complexes of terbium with small organic ligands and proteins

Oxygen does not quench the luminescence of either free Tb or of Tb bound to dipicolinate. However, sensitized Tb luminescence in complexes of that ion with elastase, thermolysin, and alpha-amylase is quenched by oxygen at rates that far exceed that with which the intrinsic fluorescence of the proteins is quenched. We infer that this more rapid quenching of Tb luminescence indicates a major role for energy transfer from tryptophan moieties in a triplet excited state.     

Analysis of the requirements for parathyroid hormone action in renal membranes with the use of inhibiting analogues.

Two synthetic analogues of bovine parathyroid hormone (PTH) with NH2-terminal modifications, PTH-(3-34) and [desamino-Ala-1]PTH-(1-34), were found to lack agonist activity but to demonstrate antagonist properties when tested in the rat renal cortical adenylyl cyclase assay in vitro against the native hormone or against PTH-(1-34), the active synthetic NH2-terminal tetratriacontapeptide. The inhibition exhibited by these analogues was proportional in degree to the dose of inhibitor, abolished by oxidation of the analogue, reversible by addition of an excess of active hormone, and specific for parathyroid hormone-stimulated renal adenylyl cyclase. No inhibition of basal or sodium fluoride-stimulated renal adenylyl cyclase could be demonstrated. Two other synthetic bovine analogues, PTH-(13-34) and PTH-(1-26), were devoid of agonist and antagonist properties. The over-all results suggest that the requirements for receptor binding of parathyroid hormone are rather broad. Conformational factors or binding interactions involving specific residues, or both seem to require the entire sequence from residue 3 to residue 27 for receptor binding to occur. A dichotomy between receptor binding and adenylyl cyclase activation was demonstrated only by alterations or deletions involving the first 2 NH2-terminal residues of the hormone and emphasizes the importance of these residues in eliciting the biological activity of parathyroid hormone. The two antagonists, [desamino-Ala-1]PTH-(1-34) and PTH-(3-34), should be useful in further analysis of the initial steps in hormone action.     

Fabry's disease as an -galactosidosis: evidence for an -configuration in trihexosyl ceramide

Pure α-galactosidases, devoid of β-galactosidase activity, were purified from coffee beans, ficin (a crude extract from figs), rat and rabbit small intestine. With the exception of the coffee bean enzyme, all α-galactosidase preparations released galactose from ³H- or ¹⁴C-labeled trihexosyl ceramide obtained from patients with Fabry's disease. Galactose liberation was specifically inhibited by α-galactosides, such as melibiose and stachyose, while lactose had no effect. Our results corroborate the α-galactosidase deficiency reported in Fabry's disease and establish that the terminal galactosyl residue of the trihexosyl ceramide stored in this condition has an α-configuration.     

Biochemical evidence for expression of a semi-allogeneic, H-2 antigen by a murine adenocarcinoma

LT-85 is an alveolegenic adenocarcinoma induced in mutant C3HfB/HeN (C3Hf) mice. This tumor, however, grows preferentially in allogeneic, wild-type C3H/HeN (C3H) mice. The tumor-associated transplantation antigen has been mapped to the K end of the major histocompatibility complex. H-2K antigens were isolated from detergent extracts of LT-85 cells by immunoprecipitation with monoclonal antibody. The tryptic peptides of these antigens were compared, by using high-pressure liquid chromatography, with the tryptic peptides of H-2K antigens isolated from syngeneic mutant C3Hf and ancestral wild-type C3H spleen cells. We found that the H-2K antigens of the LT-85 tumor cells were very similar to, but distinct from, those present on syngeneic C3Hf lymphoid cells. We also found, however, that the H-2K antigens of LT-85 tumor cells were clearly different from the H-2K antigens of allogeneic C3H spleen cells. The H-2K antigens of LT-85 cells are therefore foreign to syngeneic C3Hf cells, but do not represent expression by the tumor cells of the allogeneic H-2K antigens expressed by normal C3H cells. Furthermore, the nature of the differences observed between the H-2K antigens of LT-85 cells and C3Hf and C3H spleen cells strongly suggests that the structure of the H-2K molecule of LT-85 cells is identical in some regions to the H-2K molecule of C3Hf cells, and in other regions to the H-2K molecule of C3H cells.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

Studies of the T1 and T2 of intracellular water as a function of frequency in normal and transformed fetal cells

Frequency-dependent values of the spin-lattice relaxation time (T₁) and the spin-spin relaxation time (T₂) have been obtained for intracellular water in normal and transformed Syrian hamster fetal fibroblasts. Values of T₁ and T₂ were obtained for normal and transformed cells at 24.3 (0.57 T), 100 (2.4 T), 300 (7.0 T), and 400 MHz (9.4 T). At each frequency, values of T₁ were the same for both normal and transformed cells, whereas values of T₂ were lower for one passage of transformed cells. As expected, T₁ increased with frequency. However, T₂ decreased with frequency for both normal and transformed cells. The frequency dependence of T₂, was similar for all cells; thus, the ability of T₂ to make a distinction between normal and transformed cells did not change with field.