Fatty-acid synthesis in lactating-goat mammary gland. 1. Medium-chain fatty-acid synthesis.

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.     

Translational control of immunoglobulin synthesis. I. Repression of heavy chain synthesis

When myeloma cells are incubated at 25 °C the secretion of myeloma protein ceases within 20 minutes. The synthesis of heavy and light chains and the assembly into the completed 7 S immunoglobulin continue at over 40% of the synthetic rate at 37 °C, resulting in an increasing intracellular concentration of myeloma protein with time. When myeloma cells containing an increased myeloma protein pool were re-incubated at 37 °C, there was an initially decreased synthesis of H-chain² relative to L-chain or total protein. Whereas L-chain synthesis returned to the pre-25 °C synthetic rate within 15 minutes, the synthesis of H-chain required over 60 minutes to return to the pre-incubation rate.Myeloma cells maintained in exponential growth contain a larger intracellular pool of H₂L₂ than cells in late stationary phase. When both populations of cells were incubated at 25 °C and the synthesis of H and L-chain protein measured, a reduced synthesis of H-chain was again observed. Exponentially growing cells showed an 80% reduction of H-chain synthesis after 100 minutes at 25 °C. Stationary cells, with the reduced intracellular level of H₂L₂, required 210 minutes to effect an equivalent reduction of H-chain synthesis.The opposite effect on myeloma protein synthesis was observed following depletion of the H₂L₂ pool. The intracellular H₂L₂ pool was reduced by allowing secretion in the absence of protein synthesis. When protein synthesis was allowed to continue following the depletion, a stimulation of myeloma protein synthesis relative to total protein synthesis was observed.These experiments suggest a close relation between the intracellular level of H₂L₂ and the production of H-chain. From the rapidity of the repression and de-repression of H-chain synthesis, a regulation at the translational level is suggested.     

Histone synthesis in Rhynchosciara salivary glands. I. Site and timing of synthesis.

The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3-4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'.     

Enzymatic synthesis of malonaldehyde.

Malonaldehyde was prepared from 1,3-propanediol by alcohol dehydrogenase. The K_m for 1,3-propanediol was about 1.7 mM. The reaction proceeded best at low ionic strength and at pH 9. The reaction was unaffected by pyrophosphate, phosphate, bicarbonate, or N-ethylmorpholine buffers, or by Mg⁺², Ca⁺², EDTA, or citrate. However, the reaction was inhibited 50% by 1.5 mM borate, 1 mM cyanide, and 5 mM azide. Thiols, such as dithioerythritol, inhibited the reaction 50% at 50–100 μM, while others, such as mercaptoacetate, inhibited 50% at concentrations over 1 mM. Malonaldehyde was removed from the reaction mixture by evaporation at pH 3 and condensation at ?78°C. No other products associated with lipid peroxidation were produced. The method was useful for preparation of radiolabeled malonaldehyde.     

Combinatorial synthesis of carbohydrates.

Combinatorial chemistry principles have been applied to the generation of oligosaccharide libraries, both in solution and on the solid phase, with a view to producing inhibitors of carbohydrate-protein binding. The rich stereochemistry and high degree of functionalization of sugars has also resulted in their increasing use in the synthesis of glycomimetics and as scaffolds for the presentation of pharmacophore groupings to receptors that are noncarbohydrate-recognizing proteins.     

Inhibition of hyaluronate synthesis.

UDP-GlcNAc (UDP-N-acetylglucosamine) and UDP-GlcA (UDP-glucuronic acid) were oxidized by periodate in the ribose ring and utilized as inhibitors for hyaluronate synthase in membrane fractions from the B6 cell line and in cell cultures of B6 cells and human fibrosarcoma HT 1080. Inhibition was irreversible and concentration-dependent and could be prevented in the case of periodate-oxidized UDP-GlcNAc by UDP-GlcNAc. Periodate-oxidized UDP-GlcNAc was shown to block chain elongation of hyaluronate. Introduction of periodate-oxidized UDP-GlcA into B6 cells by hypo-osmotic lysis of pinocytotic vesicles decreased hyaluronate synthesis by direct inhibition of the synthase. In HT 1080 cells the synthesis of hyaluronate, chondroitin sulphate and heparan sulphate was inhibited simultaneously.     

Inhibition of protein synthesis also inhibits synthesis of lipid-linked oligosaccharides.

Studies were initiated to determine whether the formation of lipid-linked oligosaccharides was coupled to the synthesis of protein. Canine kidney cells were grown with [2-3H]mannose or [3H]leucine in the presence of cycloheximide or puromycin and the effect of these inhibitors on the synthesis of proteins and lipid-linked oligosaccharides was measured. In all cases, the inhibition of protein synthesis resulted in a substantial inhibition in the incorporation of mannose into the lipid-linked oligosaccharides, although the synthesis of mannosyl-phosphoryl-dolichol was only slightly inhibited. Cycloheximide had no effect on the in vitro incorporation of mannose into lipid-linked oligosaccharides when GDP-[14C]mannose was incubated with aorta microsomal preparations. The inhibition of lipid-linked oligosaccharides was apparently not due to a decrease in the amount of glycosyltransferases as a result of protein degradation in the absence of protein synthesis, nor was it the result of a more rapid degradation of lipid-linked oligosaccharides. The inhibition also did not appear to be due to limitations in the available dolichyl-phosphate. The results suggest that the formation of lipid-linked oligosaccharides may be regulated by end product inhibition.     

Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Deoxyribo-oligonucleotide synthesis*

An enzyme, which is probably identical with polynucleotide phosphorylase, was prepared from Escherichiacoli B. In the presence of Mn(2+) it catalyzes the addition of one (and to a slight extent more) residue of deoxyribonucleotide residue from the diphosphate to an oligodeoxyribonucleotide primer. The shortest effective primers contained three phosphate residues. Ribodinucleotides were effective as primers and accepted two or three deoxyribonucleotide residues under these conditions. The application of the procedures to the convenient synthesis of certain defined oligodeoxyribonucleotides up to nine residues long is discussed.     

Protein synthesis rhythm.

The three-dimensional structure of a protein molecule appears to depend on the amino acid sequence of the protein in an as yet incompletely described manner. If the amino acid sequence is replaced by a numerical sequence of values representing a physical or chemical property of amino acids, the resulting numerical sequence is amenable to autocorrelation analysis. Further, if certain geometrical parameters are calculated from the three-dimensional structure of a protein to form a configurational series, pairs of property series and configurational series can be analyzed by cross-correlation techniques. The data base for the analysis was the three-dimensional structures of ten proteins as determined by X-ray crystallography. Such analysis yields the result that the hydrophobicity of an amino acid residue in a protein influences the orientation angle of the amino acid side chain. This result is consistent with the widely current “oil-drop” model of protein structure. Hydrophobicity also appears to influence the backbone dihedral angle φ, but not ψ Such a directional effect cannot be explained by a current model of information transfer in protein helices. The magnitude of the cross correlations does not appear to be satisfactory for construction of a transfer function model for the prediction of general features of protein structure from amino acid sequences.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.