Don Dennis

Messenger RNA Structure: Compatibility of Hairpin Loops with Protein Sequence

Examination of the amino acid sequences of human cytochrome c and the α-chain variant of human hemoglobin Constant Spring has revealed the possiblity for base-paired hairpin loops in the messenger RNAs for these proteins. A similar analysis of the bacteriophage R17 coat protein suggests an additional unobserved loop in the R17 RNA. If such loops are present in messenger RNAs generally, it would suggest that DNA has more than one stable base-paired conformation.

Studies on the toxicity and antileukemic action of 6-mercaptopurine in mice.

It has been observed that 6-mercaptopurine acts as a purine antagonist for Lactobacillus casei?V2 The free purines adenine, guanine, xanthine, and hypoxanthine blocked the inhibitory activity of 6-mercaptopurine for this organism in a competitive manner; 6-mercaptopurine has also been demonstrated to have an inhibitory effect on the growth of experimental tumors and human neoplash?-? However, as in the case of other known antineoplastic agents, the toxicity of the drug for the host is a limiting factor in its employment in the treatment of neoplasia. Although the triad of host-parasite-drug has long been recognized and treated in quantitative fashion in infection chemotherapy,8-1° there has been relatively little emphasis on the host-tumor-drug relationship in tumor chemotherapy. Emphasis has been placed, in our laboratory, on the development of qualitative experimental procedures for the study of the host-tumor-drug relationship. It was felt that such procedures could provide a more firm basis for evaluation of drug effectiveness and could provide additional means for study of the mode of action of drugs. Employing citrovorum factor, folic acid, and aminopterin, experimental procedures were employed which indicate that, in the mouse, the analysis of dose-response relationships may provide a basis for inhibition analysis.’l* In addition, a macrobiological assay procedure was developed which provides a quantitative description of the antineoplastic specificity of action of a drug in terms of its relative effect against the tumor and the This procedure permits the comparison of the relative antitumor specificity of action of different treatments with the same drug, as well as of different drugs. Employing these procedures, studies were undertaken on the interrelationships of host, tumor, and drug, employing 6-mercaptopurine in mice.

LACTIC ACID RACEMIZATION

Abstract All nucleotide sequences consistent with the amino acid sequence of the α-chain of human hemoglobin were tested for their complementarity with a known 26-nucleotide sequence from the α-chain messenger. The region with the highest pairing potential is immediately adjacent to the known nucleotide sequence. The existence of this potential hairpin loop requires the specification of nucleotides in at least 6 degenerate positions.

Lactate racemase hydroxylamine-dependent 18O exchange of the α-hydroxyl of lactic acid

It has been suggested that lactic acid racemization in bacteria occurs by one of the following general mechanisms: (a) A coupled system composed of two lactic acid dehydrogenases, each specific for one of the isomeric lactic acids (e.g., Lactobacillus plantarum). (b) A single enzyme that does not function as a dehydrogenase for either isomeric lactic acid and has no detectable intermediate tha t is dissociable from the enzyme (e.g., Clostridium butylicum). Results are presented concerning the crystallization, physical and chemical characteristics of the specific dehydrogenases derived from L . plantarum. Recent studies of the mechanism of action of the racemase enzyme derived from C1. butylicum are described.

RNA Polymerase: a model for rotational translocation

Abstract The lactic acid racemase (EC 5.1.2.1) derived from Clostridium butylicum catalyzes the racemization of the α- 18 O label. The proposed α-carbonyl intermediate for the enzyme-catalyzed reaction has been previously shown to be trapped as an enzyme-bound oxime in the presence of hydroxylamine. This report demonstrates that the formation of the inactive enzyme-bound oxime, followed by reactivation in the presence of an excess of competing free carbonyl (pyruvic acid) results in a complete loss of the α- 18 O label from an original α- 18 O-labeled lactic acid.

Asymmetric phospholipid bilayer membranes: Formation and electrical characterization

DNA-dependent RNA polymerase is the enzyme involved in the transcription process. During transcription, ribonucleoside triphosphate (NTP) monomers are polymerized into a transcript (RNA) with sequence and length specified by a template DNA molecule. (ii) cyclic compound bis 3’-+5’ di-ribose 5’-phosphate (fig. 1). Functional symmetry the translocation of the growing transcript from one registry to the next is accomplished in 180” rotational increments of the enzyme in its downstream course along the DNA template. Occupancy alternates between each of the symmetrically equivalent active sites. This transcription process has two chemical mechanistic aspects: (i) The selection of the proper NTP as a substrate; (ii) The catalysis of the formation of a phosphodiester bond between the 3’-hydroxyl group of the ribose moiety at the terminus of the growing chain and the 5’ a-phosphate of the incoming NTP monomer. In the following discussion we present the topography of the active site and the dynamics of translocation.

RNA polymerase: potent competitive inhibition by D-ribose-5-triphosphate and other pentose polyphosphates.

Abstract Asymmetric bipolar phospholipid membranes were formed by juxta-position of two distinct lipid monolayers sharing a common interfacial phase. The resulting bimolecular films were stable for periods of up to two hours and displayed both normal and anomalous rectification behavior. Both membrane stability and rectification were dependent on the concentration of supporting electrolyte in the aqueous phases. Studies with calcium indicated it played an important role in determining the I–V characteristic of the film. The Katchalsky theory for membrane elements arrayed in series adequately described the systems electrical response.

Substrate concentration dependence of deuterium isotope effects on beef heart lactate dehydrogenase.

Abstract Ribose and deoxyribose 5′ pyro and triphosphates are shown to be potent substrate competitive inhibitors for an in vitro transcription system containing either calf thymus or T7 DNA as template, and the E. coli RNA polymerase [E.C.2.7.7.6]. Each of these analogues gave Ki values (∼25 μM) essentially the same as the Km values (∼15 μM) for the substrates. In contrast the ribose and deoxyribose 5′ monophosphates, ribonucleosides, deoxynucleoside mono and triphosphates were not significant inhibitors. The data are consistent with the interpretation that the enzyme binds the substrate primarily through the 3′-endo ribose polyphosphate moiety.

The use of specific macromolecular complexing agents for enzyme purification: Beef heart dehydregenase

Abstract The kinetic effect of substituting deuterium for the transferred hydrogen at the C-2 position of L-lactic acid was studied by measuring the initial velocity (v i ) of lactate oxidation by beef-heart lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27).v i H /v i D values were found to decrease with increasing substrate concentration. At saturating levels of substrate V M H /V M D was equal to one. This is an example of the masking of an isotope effect by a slow step in the reaction sequence which is not affected by deuterium substitution. Such a result is evidence that the covalent transfer of hydrogen is not the rate-limiting step in lactate oxidation. Measurement of v i H /v i D at low substrate concentration gives an estimate of the importance of steps known to be affected by deuterium substitution in the overall rate expression.