Biotin-binding Protein Research Articles

Acetyl Coenzyme A Carboxylase: III. FURTHER STUDIES ON THE RELATION OF CATALYTIC ACTIVITY TO POLYMERIC STATE

Abstract Catalytic activity of the filamentous polymeric form of avian liver acetyl-CoA carboxylase, (s20,w = 45 to 47 S) undergoes first-order decay in assay reaction mixture (t½ ∼ 10 min at 2°), which is rapidly reversed (l10 s) by addition of tricarboxylic acid activator, i.e. citrate or isocitrate. As previously shown by sucrose density gradient centrifugation, extended exposure to assay reaction mixture leads to the formation of a catalytically inactive 13 to 15 S protomeric species of the enzyme. If avidin, the biotin-binding protein from egg white, is added following this decay, reactivation by citrate is blocked completely. However, when citrate is present in the assay reaction mixture, thereby maintaining the enzyme in the polymeric state, inactivation by avidin is prevented. The presence of avidin in the assay reaction mixture does not alter the t½ for activity decay, although reactivation is no longer possible upon addition of citrate. Only that fraction of carboxylase activity remaining can be protected by citrate from further erosion of activity and inactivation by avidin. Thus, the characteristics and kinetics of the decay of catalytic activity are compatible with a transition from an avidin-insensitive, active polymeric species to an avidin-sensitive, catalytically inactive form. Consistent with a polymer (intrinsic viscosity, 83) to protomer (intrinsic viscosity, 11.5) transition, the decay of enzyme activity is tightly coupled to an apparent first order (t½ = 10 min) decline in viscosity, t½ (activity) = t½ (viscosity). Citrate addition results in the rapid restoration of carboxylase activity and an abrupt rise in viscosity, equivalent to the percentage of reactivation. Tight coupling of reversible loss of enzyme activity to depolymerization is further supported by the observation that Mg2+ ATP and HCO3-, which carboxylate the enzyme, promote a rapid loss of catalytic activity (t½ = ½ min), the formation of the avidin-sensitive species, as well as, a rapid decrease in viscosity. The presence of carboxyl acceptor (acetyl-CoA), which retards activity decay (t½ = 7½ min), and susceptibility to avidin, also delays the fall in viscosity. It is proposed that the loss of catalytic activity and formation of the avidin-sensitive species reflects a polymer to protomer transition.

Fatty Acid Synthesis by Extracts of Euglena

At least 3 known pathways are available to a cell to synithesize satuirated fatty acids: A) the malonylCoA pathwav which has been shown to result in do novo synthesis of long-chain fatty acids, B) reversal of the fl-oxidation pathway which results primarily in syinthesis of short-chain acids and onlv negligible amounlits of long-chain fatty acids. and C) a combination of these 2 processes by which lprodutcts of the malonyl-CoA system may be elongated presumably by enzymes of the :-oxidation pathway. Distinctive differences exist between these pathways for fatty acid synthesis. They differ both in the nature of the products formed and in the cofactors required when acetyl-CoA is the stubstrate. \Vhereas ATP, HCO3-, Mn+ and TPNH are required in the malonyl-CoA system only TPNH and DPNH are required for reversal of the f-oxidation pathway. Both of these systems have been wvell sttudied in animal tissue, bacteria, and yeast but to a less extent in higher plants. Very little investigation has been done with algae despite the fact that it has long been recognized that these organisms have a diverse spectrtum of both saturated and utnsaturated fatty acids and that the amotunit of unsaturation is somewhat dependent upon cutltture unlder photosynthetic conditions (12.28). In ouir past studies with extracts of the alga Enuglenci (10,11), evidence has been presented showing the requirement for ATP, Mg+, DPNH, and TPNH for incorporation of acetyl-CoA into longchain fatty acids. Stoichiometric studies have given results analogous to those obtained in the malonylCoA systemn, i.e., 1 ATP and 2 reduced pyridine nucleotides are tutilized per acetyl-CoA incorporate(d into fatty acids (60-70 % of 16-20 carbon atoims). Bicarbonate wlhiclh is essential for fatty acid synithesis via the imalonyl-CoA pathway is, however, not required, and the biotin-binding protein avidin is not inhibitory. These observations and the fact that the rate of incorporationi of malonyl-CoA is infinitesimally smiiall compared to that of acetyl-CoA would suggest that we are not dealing with fatty acid synthesis via the malonyl-CoA pathway. We wish to report here on further studies of the products and certain other properties of the system. These studies include the effect of avidin on distribution of products, the distribution of C'4 in stearate biosynthesized from acetyl-1-C'4-CoA, and the chemical nature of the products. In addition, we shall give data on the sensitivity of the fatty acid synthesizing system to various -SH inhibitors and the specificity of the system for nucleoside triphosphates.

The properties of streptavidin, a biotin-binding protein produced by Streptomycetes

Streptavidin, a crystalline protein isolated from fermentation filtrates of streptomycetes has been reported to have biotin-binding ability similar to avidin. A comparison has been made between streptavidin and avidin from egg white using microbiological, physical, and chemical methods. Streptavidin binds four molecules of biotin. Chemical analysis shows the absence of hexoses and amino sugars associated with avidin and reveals differences in amino acid content. Electrophoretic studies give different mobilities for streptavidin and avidin. Thus, streptavidin is a new type of biotin-binding protein.

BIOTIN AND PROPIONYL CARBOXYLASE

tracts of biotin-deficlent rat liver carboxylated propionate at a greatly reduced rate, highly purified preparations of propionyl carboxylase from pig heart were assayed for biotin;2 however, none was detected at that time. A reinvestigation of this problem was prompted by the finding that preparations of acetyl4 and A-methylcrotonyl5 carboxylase from liver and a bacterial source, respectively, contained biotin and were inactivated by avidin, the biotin-binding protein of egg white. The reactions catalyzed by these enzymes-carboxylation of acetyl CoA to malonyl CoA and of f-methylcrotonyl CoA to 3-methylglutaconyl CoA-conform to the pattern of reaction 1.6 It appeared, therefore, that propionyl carboxylase must contain