Crystallisation and preliminary X-ray data of ribulose-1,5-bisphosphate carboxylase from spinach

Crystals of a tertiary complex of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase with the activators Mg²⁺ and CO₂ have been grown. These crystals diffract strongly to 1.6 Å resolution. The spacegroup is C222₁ with unit cell dimensions a = 158.6 Å, b = 158.6 Å, c = 203.4 Å. Additional local symmetry is apparent in the pattern of absences and the intensity distribution of the X-ray precession photographs. The photographs have been interpreted in terms of a molecule (consisting of eight large and eight small subunits, L₈S₈) with 222 symmetry and a molecular centre shifted 2 Å in the x direction from the origin of the unit cell. The asymmetric unit contains half the L₈S₈ molecule. The intensity distribution suggests that the molecular symmetry does not deviate far from 422. These crystals are compared with other crystalline forms of the enzyme and the implications of these results are discussed.     

D-lysine catabolic pathway in Pseudomonas putida: interrelations with L-lysine catabolism

The isolation of several mutant strains blocked in l-lysine degradation has permitted an assessment of the physiological significance of enzymatic reactions related to lysine metabolism in Pseudomonas putida. Additional studies with intact cells involved labeling of metabolic intermediates from radioactive l- or d-lysine, and patterns of enzyme induction in both wild-type and mutant strains. These studies lead to the conclusions that from l-lysine, the obligatory pathway is via delta-aminovaleramide, delta-aminovalerate, glutaric semialdehyde, and glutarate, and that no alternative pathways from l-lysine exist in our strain. A distinct pathway from d-lysine proceeds via Delta(1)-piperideine-2-carboxylate, l-pipecolate, and Delta(1)-piperideine-6-carboxylate (alpha-aminoadipic semialdehyde). The two pathways are independent in the sense that certain mutants, unable to grow on l-lysine, grow at wild-type rates of d-lysine, utilizing the same intermediates as the wild type, as inferred from labeling studies. This finding implies that lysine racemase in our strain, while detectable in cell extracts, is not physiologically functional in intact cells at a rate that would permit growth of mutants blocked in the l-lysine pathway. Pipecolate oxidase, a d-lysine-related enzyme, is induced by d-lysine and less efficiently by l-lysine. Aminooxyacetate virtually abolishes the inducing activity of l-lysine for this enzyme, suggesting that lysine racemase, although functionally inactive for growth purposes, may still have regulatory significance in permitting cross-induction of d-lysine-related enzymes by l-lysine, and vice versa. This finding suggests a mechanism in bacteria for maintaining regulatory patterns in pathways that may have lost their capacity to support growth. In addition, enzymatic studies are reported which implicate Delta(1)-piperideine-2-carboxylate reductase as an early step in the d-lysine pathway.     

Dehydrogenation of a phosphonate substrate analogue by glycerol 3-phosphate dehydrogenase

S-(+)-3,4-Dihydroxybutylphosphonic acid, an isosteric analogue of sn-glycerol 3-phosphate, was synthesized stereospecifically and shown to be an effective substrate for rabbit muscle glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate-NAD(+) oxidoreductase, EC 1.1.1.8). Non-isosteric phosphonate analogues of sn-glycerol 3-phosphate showed neither substrate nor inhibitory activity with the enzyme.     

Mechanical Deformability of Biological Membranes and the Sphering of the Erythrocyte

Equations of mechanical equilibrium are applied to the erythrocyte membrane in the normal, hypotonically swollen, and sphered configurations. The hydrostatic pressure drop across the normal cell membrane is shown to be zero for all biconcave shapes if the membrane thickness is uniform. This result leads to the conclusion that the membrane tension is uniform and is a function of membrane potential. A two-dimensional fluid film model for the membrane is introduced to describe the unusual deformability of the erythrocyte during sphering in hypotonic solutions. The model predicts a smooth transition from the biconcave shape to a perfect sphere.     

ACTIVATION IN VITRO OF RAT LIVER POLYRIBOSOMES

The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35°C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.