Ph.D. - Biomedical Sciences (Biochemistry)

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Studies of human serum albumin-ligand interactions using site-directed mutants and recombinant fragments of the protein
(University of Hawaii at Manoa, 2004) Yang, Jinsheng, 1961
Studies of a sulfhydryl oxidase from the male reproductive tract: a sperm-protective enzyme
(University of Hawaii at Manoa, 1976) Chang, Thomas S.K.
Studies on biochemical changes associated with in vitro capacitation in golden hamster spermatozoa
(University of Hawaii at Manoa, 1972) Rogers, Beverly Jane
Correlation between intramolecular base composition heterogeneity of DNA and control of transcriptional expression in E. coli temperate phage P2
(University of Hawaii at Manoa, 1972) Geisselsoder, Janet
Intramolecular base compositional heterogeneity has been demonstrated in the DNA of the phage P2 by following the optical density of solutions at 260 nm as a function of increasing temperature. First derivative curves of the functions thus generated have been obtained for P2 DNA and the DNA of two closely related phages. Comparison of these curves reveals similarities at the high temperature (high GC) end and differences at the low temperature (low GC) end. All three of these phages can, by supplying the genes concerned with phage particle maturation, serve as helper for the defective phage P4. The strands of P2 DNA have been separated on the basis of their buoyant densities in CsCl solutions when complexed with poly UG. In vivo transcription patterns from these strands in cells infected with various P2 mutants have demonstrated that the "heavy" strand is the one predominantly transcribed, that some "light" strand transcription originates from an operon coding for proteins involved in phage head assembly (thus fuling out a "read-through" mechanism for late gene activation), and that early "light" strand transcription does not originate from DNA deleted in two mutants which is in the right half of the physical map. There is, however, some "light" strand transcription early in infection. P2 DNA has been sheared in: half and the halves separated on the basis of Hg++ binding in Cs2SO4 density gradients. Electron microscopic analysis of partially denatured molecules in these preparations have fixed them with respect to the physical map. In ~ transcription originates primarily from the right, or low GC half early in infection and then shifts to the left, or high GC half. Mutants in genes A and B, which are located in the right half of the genetic map, are defective in both DNA replication and in effecting this shift. Infection with one polar amber mutant under non-permissive conditions has demonstrated that this operon lies :in the right half of the DNA and thus helps to fix the physical map with respect to the genetic map. Two of those regions of the phage DNA which are known to be transcribed from a repressed genome, namely the prophage, appear to be of quite low GC content.
The synthesis and release of hormones from human adenohypophyses in vitro
(University of Hawaii at Manoa, 1971) Siler, Theresa Marie
Isolation of human prolactin (HP) from fetal and tumor culture medium which was distinct from human growth hormone was achieved by column chromatography. These preparations were studied by chromatoelectrophoresis, radioimmunoassay and polyacrylamide gel electrophoresis (PAGE). The isolated fractions were then compared with two preparations of Pasteels' human prolactin which were further purified by Dr. Lyons (Lyons' PHP and Lyons' cPHP) and the other HP preparations by radioimmunoassay, PAGE and NH2-terminal studies. The HP preparation from fetal culture medium was found to be the most highly purified and immunopotent preparation and most suitable for a radioimmunoassay for human prolactin in plasma. Forty-four human fetal pituitaries and two human pituitary adenomas were studied in organ culture for up to 11 months. Radioimmunoassays for human growth hormone (HGH), human prolactin (HP), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), adrenocorticotropin hormone (ACTH) and melanocyte-stimulating hormone (MSH) were performed on the culture medium from the pituitary organ cultures. The hormones released into the medium were studied throughout the time in culture and related to the age of the fetal pituitary when first cultured and to its sex. The release of HGH, FSH, LH and ACTH gradually decreased during the first 20 days in vitro to basal or undetectable levels. The release of HP and MSH initially decreased and then increased with the time in culture to a sustained elevated level of release. The total release of each hormone was studied and could be related to various events in fetal development. Pituitaries from six fetuses which had an abnormal total release of pituitary hormones could be related to a fetal abnormality in three cases. Studies of pituitary anlage tissue from embryos as early as five weeks of gestation released hormones in vitro. That these hormones were synthesized in vitro was shown by incorporation of Cl4-leucine into these hormones as determined by PAGE and specific antibody precipitation of each hormone prior to PAGE. Some factors controlling the release of hormones in vitro were studied by the use of modified media. An increased release was obtained with an increased Ca++ and K+ in the medium whereas EDTA caused a decrease in FSH and LH and an increase in HGH and HP. With increased Mg++ concentrations in the medium HGH, ACTH, and HP release decreased. The removal of estradiol from the medium results in increased release of FSH, LH, HGH and ACTH while the HP release was greatly decreased. Various doses of TRH decreased HP release and altered the release pattern of HGH, FSH, and LH. Fetal hypothalamic tissue added to the medium increased HGH, FSH and LH release and extended the duration of release for these hormones while HP release was inhibited. Chick embryo extract was found to have a stimulating effect on the release of each hormones in vitro.
Investigation of manganese binding potential in neoplastic and non-neoplastic tissue
(University of Hawaii at Manoa, 1971) Zimmerman, Norman
In an attempt to discover methods of controlling cancer, two basic philosophies of research prevail. The first involves testing vast multitudes of drugs in the hope that one may be found which destroys the malignancy without killing the patient. The second espouses the finding of differences between neoplastic and non-neoplastic tissue in the hope that these differences may elucidate the nature of the carcinogenic process. Once this is known, one would hope to be able, teleologically, to select possible methods of cure. Clearly, a great number of differences between neoplastic tissue and the tissue of origin of the neoplasm simply reflect cellular differences and in no way relate to the carcinogenic process. The dissertation will be concerned with the differences in manganese binding potential in neoplastic and non-neoplastic tissue that has been observed by the use of Electron Paramagnetic Resonance (EPR). A general discussion of EPR and the role of manganese in normal tissue will be followed by a proposed theory of carcinogenesis. Evidence will then be presented for differences in manganese binding potential between neoplastic tissue and the tissue of origin for six different systems. Competition studies will be utilized to show that the effect observed with manganese cannot be seen for a variety of other ions (i.e., it is not a common ion effect). A brief discussion on endogenous manganese levels will be included. A discussion of purification of the binding system will follow. In conclusion, data will be presented regarding manganese binding potential with respect to the carcinogenic process.
The pyrimidine tracts of bacteriophage ΦX174
(University of Hawaii at Manoa, 1969) Bickel, Riva Pearl Wenig
The bacteriophage ϕX174t containing single stranded, circular DNA as its genetic material, has been studied in some detail with respect to its chemistry and biological function. The frequency of the pyrimidine tracts in the wild type bacteriophage has been determined t and the preponderance of the amount of longer tracts of seven to ten pyrimidine nucleotides, over that which would be expected from a random distribution noted. Further studies of the longer pyrimidine tracts were carried out using the ρ- mutant bacteriophage, which is lysis defective, showing delayed lysis in dilute cultures of the host, E. coli, and lysing only about ten per cent of the cells in dense culture. The DNA of the phage contains a discontinuity, a small region which is not single stranded, and therefore not susceptible to the action of E. coli exonuclease I. This enzyme digests only single stranded DNA and works only on free 5' OH ends. Therefore, digesting ϕX174 chromosomes with pancreatic DNA'ase, an endonuclease, to a limited extent, followed by digestion with E. coli exonuclease I, leaves a population of DNA of varying lengths, all having the same 5' end at the discontinuity. Within this population, then, the frequency of an individual tract's survival, depends on its distance from the discontinuity, and can be used to determine its position on the ϕX174 chromosome. Methods for preparation of pyrimidine tracts, their separation by length into isopliths, and their subfractionation according to their relative cytosine and thymine content have already been developed. When more than one tract of the same length and base composition existed, further separations were not possible. Only the unique tracts could be mapped. This, in the ρ- mutant, consisted of one decanucleotide, two each of nona and octanucleotides and one each of hepta and hexanucleotides. All of these tracts were located in the region of about 30 to 80 per cent of the distance around the chromosome from the discontinuity. The pyrimidine tracts of the ρ- mutant and wild type bacteriophage were compared. There is a decanucleotide of composition C3T7 present in the wild type phage, but not in the ρ- mutant. Some evidence indicates that this mutation, that of a pyrimidine to a purine, occurs at a cytosine residue, leaving an octanucleotide, CIT7 present in the ρ- mutant, but not in the wild type phage. The nature of the ρ- mutation is discussed, along with the possibility that the longer pyrimidine tracts may have a role in initiation of the DNA to messenger RNA transcription process.
A study of the biosynthesis of growth hormone and prolactin in bovine pituitary slices and cell-free systems
(University of Hawaii at Manoa, 1969) Robertson, Mary Chalmers
The biosynthesis of growth hormone and prolactin was studied in both slice and cell-free systems from bovine anterior pituitary tissue. Slice preparations were highly active in incorporating radioactive amino acids into prolactin t growth hormone and protein. Modification of existing procedures were employed for the isolation of the labeled hormones in a relatively purified state. A number of physical t chemical, and biological criteria. were used to demonstrate that the radioactive proteins were indistinguishable from reference standards. These included amino acid composition, N- and C-terminal analysis, gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and Ouchterlony immunodiffusion. Using the same procedure, the relationship of growth hormone releasing factor (GRF) to the synthesis and release of growth hormone in vitro with both bovine and rat pituitaries was studied. Although no effect on synthesis and only a slight effect on release were noted, these results must be considered inconclusive because of the small number of samples studied. With a cell-free system, consisting of ribosomes plus pH 5 enzyme fraction, and fortified with ATP, GTP, and Mg++, the optimal conditions for biosynthesis of growth hormone and general protein were established. When a polysome-enriched preparation was resolved into fractions of discrete particle size on sucrose density gradients, incorporation of radioactive amino acids into the hormone was most active with polysomes containing six to seven ribosomal units. The isolated radioactive product was indistinguishable from the authentic growth hormone standard by four physical criteria; ion-exchange chromatography, Sephadex gel filtration, polyacrylamide disc gel electrophoresis and sucrose density gradient centrifugation. In addition, it displayed specific antibody binding. Examination by similar physical criteria, of the radioactive polypeptide associated with the prolactin fraction isolated from the pituitary ribosomal system, revealed no evidence of incorporation of labeled amino acids into the monomer form of prolactin. Several explanations have been offered for the failure to demonstrate synthesis of prolactin with this cell-free system.
The effect of steroids on RNA synthesis in various tissues of the rat
(University of Hawaii at Manoa, 1969) Philleo, William Wallace
Single doses of aldosterone were administered to normal rats, and the effect of RNA synthesis was studied. The maximal stimulation of the incorporation of l4c-ATP into RNA by nuclei isolated from rat kidneys was observed 30 minutes after an intravenous injection of aldosterone. Thereafter, the RNA synthesis decreased to a level less than that of the control, reaching a minimum at 2.5 hours. The return to control level was followed by further oscillations in RNA synthesis. Likewise, in isolated brain nuclei, an oscillation in RNA synthesis was observed following an intravenous injection of aldosterone. The effect of single injections of aldosterone, cortisol, deoxycorticosterone acetate, testosterone or progesterone was studied on RNA synthesis by isolated rat spleen nuclei. It was found that the l4C-ATP incorporation was initially inhibited following the injection of aldosterone, cortisol or deoxycorticosterone acetate. The initial response to an injection of testosterone or progesterone was a stimulation in the spleen RNA synthesis. The response to the administration of aldosterone was similar to that of cortisol. Subsequent to the initial inhibition, a stimulation followed by a return to the level of the control, was observed and secondary inhibitions were found. RNA synthesis in the thymus was initially inhibited following the administration of aldosterone. RNA synthesis was studied in the nuclei isolated from the kidney and spleen of adrenalectomized or hypophysectomized rats injected with aldosterone. Oscillations were observed in the RNA synthesis of both tissues of the endocrinectomized rats. It was concluded that the oscillations in RNA synthesis were not a function of some form of homeostatic control involving the pituitary and adrenal glands. Aldactone (SC 9420) was found to modify the RNA synthesis of nuclei isolated from the kidney, spleen and thymus of intact, adrenalectomized or hypophysectomized rats. Again, oscillations in RNA synthesis were observed, however there was the suggestion that the adrenals or the pituitary might be involved in the response as observed in the kidney and the thymus. Experiments were carried out in which both aldosterone and Aldactone were injected, and their action on RNA synthesis was observed in the kidney, spleen and thymus. The results indicated that these two steroids alter RNA synthesis independently from each other. Analysis of the RNA synthesized in vitro by the isolated nuclei of the kidney and spleen indicated that only a single species of RNA was made. This 5 s RNA was identical to that species of RNA present in nuclei, prior to incubation for the incorporation of l4C-ATP.
The behavior of horse liver alcohol dehydrogenase in guanidine hydrochloride solutions
(University of Hawaii at Manoa, 1969) Green, Robert William
The Z average molecular weight of horse liver alcohol dehydrogenase (LADH) in dilute neutral buffer was found to be 78,200 by sedimentation equilibrium. One molar guanidine hydrochloride (GuHCl) reversibly inhibited LADH activity. The inhibition was competitive with limiting nicotinamide adenine dinucleotide and mixed with limiting ethanol. When EDTA was added to LADH in 1 M GuHCl, zinc was removed followed by aggregation. Sedimentation velocity experiments indicated that no subunit was formed under these conditions. Higher concentrations of GuHCl resulted in dissociation of LADH. In 3 M GuHCl, the sedimentation equilibrium data indicated the presence of a reversible association-dissociation system involving subunit, dimer, and trimer. EDTA removed zinc but had no effect on the molecular weight of LADH in 3 M GuHCl. Reduction and alkylation of LADH in 3 M GuHCl resulted in almost complete dissociation into two subunits. Apparently, sulfhydryl residues are involved in subunit association, while zinc plays little or no role. The Z average molecular weight of LADH in 5 M GuHCl containing mercaptoethanol was found to be 45,700, a value higher than expected. The discrepancy may be attributed to incomplete dissociation, a small error in apparent partial specific volume, or solvent binding. Measurements of molecular weight, sedimentation coefficient, and intrinsic viscosity of LADH in 5 M GuHCl indicate that the structure of LADH is composed of two subunits.
The preparation of a cell free system from Bacillus subtilis capable of carrying out protein synthesis
(University of Hawaii at Manoa, 1968) Migita, Lloyd Kazuo
α-Amylases have been obtained in highly purified crystalline forms from Bacillus subtilis (Yamamoto, Bull. Agr. Chem. Soc. Japan 19, 121, 1955; Fillig et al., Helv. Chim. Acta. 40, 529, 1957; Hagihara, Proc. Japan Acad. 27, 346, 1951). A new purification method for obtaining crystalline α-amylase starting with a crude commercial enzyme preparation from the Pacific Laboratories strain B. subtilis has been developed. The purification method employs the use of chromatography on DEAE-cellulose, Duolite A-2, and hydroxylapatite. The crystalline enzyme is shown to be homogeneous by the criteria of ultracentrifugation, electrophoresis, amino terminal amino acid analysis, and rechromatography. Unlike other B. subtilis α-amylase (Stein et al., J. Biol. Chem. 232, 867, 1958) which has been reported to occur as a dimer with molecular weight of 100,000 with a zinc ion holding the subunits together, Pacific Laboratories α-amylase yielded a molecular weight value of 48,700 by the Sephadex gel filtration method. Also, the amylase was shown to contain only trace amounts of zinc (0.11 moles zinc/50,000). Other studies on the chemical and physical properties are also reported. A cell free extract capable of incorporating radioactive amino acids into hot trichloroacetic acid insoluble proteins has been prepared from the Pacific Laboratories strain B. subtilis. The endogenous activity of the subcellular system was dependent on the age of the B. subtilis culture. Thus, subcellular systems that are prepared from the exponentially growing bacteria yielded highly active systems. The amino acid incorporation is dependent upon ATP and ATP generating system, magnesium ions, ammonium ions, an amino acids mixture, and a sulfhydryl reagent. Omission of GTP did not result in a decrease of incorporating activity as reported in other B. subtilis system (Taubman et al., 1964, In Antimicrobial Agents and Chemotherapy, p. 395). The incorporation of amino acids is sensitive to ribonuclease and deoxyribonuclease which inhibited the system 92% and 20% respectively. The antibiotics puromycin (90% inhibition), chloramphenicol (31%), and mitomycin c (18% inhibition), also decreased the incorporation of amino acids. The formation of α-amylase in the subcellular fractions of B. subtilis was demonstrated by following the increase of enzyme activity with time. Increase of α-amylase activity could be demonstrated in subcellular fractions isolated from both exponentially growing cells and stationary phase cells. This is in contrast to a previous report by Oishi et al. (Biochem; Biophys. Res. Comm. 8, 342, 1962) who reported increase of enzyme activity in subcellular fractions from stationary phase cells only. The observed increase of α-amylase in the Pacific Laboratories B. subtilis system is shown to be partly due to an energy dependent system and partly to an energy independent process, presumably, the release of preformed enzyme. That part of the process is not a de novo synthesis of α-amylase is supported by the fact that addition of ribonuclease and puromycin increased the enzyme activity.
Steroid hormones and subcellular processes
(University of Hawaii at Manoa, 1968) James, Gordon Price
Aldosterone administered intraperitoneally induced an increase in the rate of renal RNA synthesis in the rat. A maximum response of 130 percent of control occurred 1.5 hours after injection. Following the maximum at 1.5 hours, the rate of renal RNA synthesis oscillated about control. Three cycles in the rate of kidney RNA synthesis occurred within 4.5 hours after injection with no indication of a decrease in amplitude. Renal RNA synthesis is stimulated to a maximum of 210 percent of control 30 minutes after an intravenous aldosterone injection. Following the maximum at 30 minutes, the rate of kidney RNA synthesis oscillated about control but with longer periods and greater amplitude than when the hormone was given intraperitoneally. Aldosterone induced oscillations in renal RNA synthesis occurred in normal, adrenalectomized and hypophysectomized rats. Aldosterone in doses of 0.07 pg to 2.5 pg was effective in inducing the oscillations. Intravenous administration of cortisol or aldosterone diminished the rate of splenic RNA synthesis in rats. Inhibition to 70 percent of control occurred within four hours after hormone injection. Following the initial inhibition, a rapid increase occurred to approximately 160 percent of control. The maximum occurred at five and six hours for cortisol and aldosterone, respectively. A rapid decrease below control level followed the stimulation. The aldosterone induced oscillations in splenic RNA synthesis was observed in normal, adrenalectomized and hypophysectomized rats. The hormone induced oscillations in renal and splenic RNA synthesis appear to be unrelated. The possibility is suggested that the oscillations are unique functions of the respective tissues and that they are independent of external control.
Beef liver mitochondrial amine oxidase: purification and studies on some physical and chemical properties
(University of Hawaii at Manoa, 1968) Gomes, Benedict
Beef (steer) liver mitochondrial amine oxidase was prepared according to the method reported earlier (Adv. Pharmacol., 6, Part A, 43, 1968). In addition to the usual preparation with high activity, (component 2, specific activity of 8,000) another component of the enzyme (component 1) with lower activity (specific activity 3,000) was isolated (Biochem. Biophys. Res. Commun., submitted). Studies were made on some physical and chemical properties of these two components. The amine oxidase components were bright yellow in color; they were thermolabile, and unstable at room temperature. The rate of inactivation of component 2 was faster than that of component 1. The optimum pH for activity was found to be 9.2. Both the components were non-competitively inhibited by p-chloromercuribenzoate. Metal chelators like cuprizone, 8-hydroxyquinoloine, o-phenanthroline inhibited the enzyme components. Ammonia or aldehyde reagents did not have significant effects on the activity. Both the components had almost the same substrate specificity. The molecular weights of the enzyme component 1 was found to be 400,000 by the gel filtration technique, 396,000 ± 10,000 on the basis of Stoke's radius, sedimentation coefficient, and partial specific volume, and 425,000 ±10,000 on the basis of sedimentation diffusion method. These values for component 2 were 1,300,000, 1,195,000, and 1,355,000, respectively. The sedimentation coefficients of component 1 and component 2 were 14.4 ± 0.3 and 20.6, respectively. Metal analyses of the enzyme yielded 1 gram atom of copper per 400,000 grams or 3 gram atoms of the metal per mole of component 2. Other metals, such as cobalt, iron, manganese, and molybdenum were examined and found to be either absent or insignificant (J. Biol. Chem., 241, 2774, 1966). Both the components of the mitochondrial enzyme were found to be flavoproteins. This was amply proved (1) from their riboflavin content as determined microbiologically, and spectrophotometrically (Biochem. Biophys. Res. Commun., 23, 324, 1966), (2) from a steady increase of riboflavin during purification processes, and (3) from the spectrum of flavo-peptide obtained from pronase digest of the enzyme. Besides, the prosthetic group was found to contain ribose (Biochem. Biophys. Res. Commun., 29, 562, 1967), adenine and phosphorus in integral values suggesting that the "flavin prosthetic" group was a flavin adenine dinucleotide of unknown structure. Accordingly, component 1 contained 4 and component 2 contained 12 FAD or FAD-like substance per mole, respectively. Examination of the sulfhydryl groups revealed that components 1 and 2 of the enzyme contained 28 and 86 titratable sulfhydryl residues, respectively in their molecules, and that they were not directly involved in enzyme catalysis. In addition, the enzyme was found to contain 24 and 106 moles of phospholipid in components 1 and 2, respectively. Finally, it appeared that the high molecular weight component was the native form from which the small molecular component arose during the purification of the enzyme, although no interconversions were observed with the purified enzyme preparations.
Multiple forms of bacterial hydrogenases
(University of Hawaii at Manoa, 1968) Asato, Robert Noriyoshi
Hydrogenase enzymes from many distinct bacterial species have been shown by disc electrophoresis on polyacrylamide gel to exist in multiple forms. The enzymes of the hydrogenase systems have different electrophoretic mobilities and produce a band pattern that is unique for each species. These studies have demonstrated the utility of this method for the taxonomic identification of hydrogenase containing bacteria. A tentative identification of an isolated bioluminescent bacteria as P. phosphorium was made using this method. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (RF) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, iron in the medium, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band patterns. The specificities of various isoenzymes of certain bacteria were tested with redox dyes of different potentials, and no difference among isoenzymes of a particular organism was observed. Three of the six forms of C. pasteurianum were found to be interrelated with the ∝ and β forms always converting to the more stable y species. The molecular weights of these forms were approximately 50,000. Recently developed techniques such as sucrose density gradient centrifugation, and gel filtration allowed studies on hydrogenase in the presence of other proteins by a direct assay of its biological activity. Comparative studies were done on the size and shape of hydrogenase enzymes of C. butyricum and C. butylicum, with the former possessing isoenzymes of the same weight as those of C. pasteurianum. Kinetic studies on purified preparations of C. pasteurianum and C. butylicum hydrogenases demonstrated that molybdenum was involved in the one-electron transfer to methyl viologen. The participation of iron in the viologen assay of C. butylicum hydrogenase was also shown. A purification scheme for the isolation of C. butylicum hydrogenase is presented. The final extract possessed a specific activity of approximately 4.Z liters HZ evolved per hour per mg protein. This represented a 500 fold purification over the crude preparation and was stable when stored under HZ at 0° for up to several weeks. The estimation of the turnover number of this partially purified form of hydrogenase indicate that it is at least three times more active than catalase, the most active enzyme known. It has been shown that for all hydrogenase enzymes examined there is an abrupt change in the Arrhenius activation energy at 17°. This was true for a variety of hydrogenase enzymes, catalyzing a variety of reactions. This transition temperature corresponds exactly with a similar transition in the electrophoretic mobilities of the various hydrogenases on polyacrylamide gel. It is believed that this phenomenon is the manifestation of a conformational change occurring at this temperature.
Isolation and characterization of taro ferredoxin
(University of Hawaii at Manoa, 1968) Rao, K. Krishna
Characterization of bacterial hydrogenase
(University of Hawaii at Manoa, 1968) Kidman, Antony David
Hydrogenase, an enzyme found in microorganisms, plays an important role in the disposition of electrons during anaerobic metabolism (Grey and Gest, Science 148, 186, 1965) and has been implicated in nitrogen fixation (Lee and Wilson, J. Biol. Chem. 151, 377, 1943). Comparative studies on the physical properties have been impossible to date because of the enzyme's instability upon purification (Sadana and Jagannathan, Biochim. Biophys. Acta 19, 440, 1956). Recent investigations by Ackrell et al. using direct assay with a redox dye on acrylamide disc gels (J. Bacteriol. 92, 828, 1966) have indicated the presence of hydrogenase isoenzyrnes in a wide variety of microorganisms. In the present work three isoenzymes of C. pasteurianum (α, β and γ) were found to possess the same molecular weights in the region of 50,000. The α and β species would convert to the more stable γ species on storage overnight. Techniques such as gel electrophoresis, sucrose density gradient centrifugation, and gel filtration permitted studies on hydrogenase in the presence of other proteins by direct assay of biological activity. Kinetic studies on C. pasteurianum hydrogenase by use of the appropriate redox dyes, methyl viologen and methylene blue, showed that molybdenum was involved in one electron transfer, but not in two electron transfer. A. vinelandii hydrogenase, believed to be involved in nitrogen fixation, was subjected to time course studies to determine the stability of the isoenzymes during storage and culture growth; no significant changes in the isoenzyme band patterns were found. Hydrogenase existed in A. vinelandii in both a soluble and particulate fraction, which could be solubilized upon appropriate treatment. The molecular size (130,000) and isoelectric point (pI =5) of the soluble and solubilized preparations were found to be similar. This observation, together with the fact that soluble hydrogenase was released by the particulate material on standing and during disruption, suggested that the two preparations contained the same enzyme. Comparative studies were performed on the size and shape of the hydrogenase isoenzymes of the following organisms. (1) The strict anaerobes: C. felsineum (59,000), C. butylicum (105,000), C. butyricum (53,000), and D. desulfuricans (56,000). (2) The facultative anaerobes: P. vulgaris (185,000) and E. coli (215,000). A. vinelandii, D. desulfuricans and the facultative anaerobes which contained cytochromes possessed both soluble and particulate hydrogenase activity. The soluble material was resolved into a complex form (mol. wt. 2-3 x 10^6) and a free form. It was thought that an equilibrium existed between these two states, and furthermore that the complex was associated with the cytochromes of these organisms. Thus the size and properties of the hydrogenase enzymes showed significant differences which depended upon the type of electron transfer process occurring in the organism.
The bovine anterior pituitary biosynthetic cell-free system: isolation and biogenesis of ACTH
(University of Hawaii at Manoa, 1968) Hussa, Robert Oscar
The association-dissociation and some physical-chemical studies of plasma amine oxidase
(University of Hawaii at Manoa, 1966) Achee, Frances Maunaalani
Beef plasma amine oxidase has been isolated and crystallized by Yamada and Yasunobu (J. Biol. Chem., 237, 1511, 1962). The high molecular weight reported for the enzyme and other preliminary studies (Gee, Master's Thesis, U. of Hawaii, 1963) indicated that the enzyme might be composed of subunits. An investigation was made of the 3ssociationdissociation properties of plasma amine oxidase. The usual methods employed to break down non-covalent linkages in proteins, such as raising or lowering the pH, detergent treatment, succinylation, and urea denaturation, proved to be unsuccessful in dissociating the enzyme as revealed by ultracentrifugal analysis of the treated protein. Dissociation was achieved only in 6 M guanidine-hydrochloride in the presence of ~ reducing agent. Determination of Sulfhydryl groups and the total cystine and cysteine content of the enzyme revealed that there are two free thiol groups and nine disulfide bonds present in the molecule. The number of -S-S- bridges gave support to the hypothesis that the polypeptide chains of PAO are covalently bonded. A re-evaluation of the molecular weight of the native enzyme led to the realization that PAO is a chemically interacting system and exists in solution as monomeric species in rapid equilibrium with higher polymers. The techniques of sedimentation equilibrium and Sephadex gel filtration gave a molecular weight of 170,000 for native PAD. The high molecular weight of 261,000 previously reported (Yamada et al., Biochim. Biophys. Acta, 8l, 165, 1964) was explained as probably being an average of the monomer and higher polymers. Molecular weight determinations of the reduced enzyme in 6 M guanidine- hydrochloride by sedimentation equilibrium and sedimentation-diffusion methods yielded values of 86,600 and 87,000, respectively. The data suggested that plasma amine oxidase is composed of two polypeptide chains which are covalently linked by disulfide bonds. The enzyme was found to undergo a loss of activity upon standing at low temperatures which was characterized by the presence of faster moving components along with the original component in the ultracentrifuge. The sedimentation coefficients of these components were indicative of the presence of monomers, dimers, and trimers. This association phenomenon appeared to be a mass action effect. A 1% enzyme solution in 0.06 M phosphate buffer, pH 7.0 gave rise to the presence of more polymer and greater loss of activity than did a 0.5% solution prepared and kept under identical conditions.
Nitrite reduction in Clostridium pasteurianum
(University of Hawaii at Manoa, 1966) Edwards, Gordon Clide
The mechanism of nitrite reduction in plants and bacteria has not been well defined. The identification of a new, low molecular weight, non-heme iron protein, ferredoxin, as an electron carrier in C. pasteurianum, led to the discovery of a closely associated nitrite reductase activity in this organism (Mortenson, L. E., et al., Biochem. and Biophys. Research Communs., 2, 448 (1962)). This nitrite reductase system was chosen for a detailed study as an example of a ferredoxin-dependent reaction. Studies with the cell-free extract from which the ferredoxin had been removed, called the phosphoroclastic enzyme system, substantiated the reaction sequence: H2-hydrogenase-ferredoxin-nitrite reductase-N02- NH3 proposed earlier (Valentine, R. C., et al., J. Biol. Chem. 238, 856 (1963)). Nitrite was stoichiometrically converted to ammonia. The six-electron reduction occurred as one complete' reaction, and no intermediates were detected. The Michaelis constant for the overall reaction, Km =9.1 µM. Experiments with reduced flavin and pyridine nucleotides added to reaction mixtures or generated In ~ demonstrated that these nucleotides do not participate in nitrite reduction in this organism. Ferredoxin apparently mediates the electron transfer by coupling directly with the nitrite reductase system. With sodium dithionite as the electron source, benzyl viologen mediated the direct supply of electrons to nitrite reductase, but not to hydrogenase. This system provided a means of isolating the nitrite reduction activity from other cellular systems and of studying it independently of hydrogenase and ferredoxin activities. Substitution of other artificial dyes for benzyl viologen indicated the enzyme could accept electrons only from sources that had electrode potentials similar to the hydrogen electrode (E'0 =-0.35 to -0.45 v at pH 7). Nitrite reductase was inhibited reversibly by CO. Established sulfhydryl and iron inhibitors were also effective inhibitors of this enzyme system. The enzyme is apparently a metalloprotein containing iron. The Km =25 µM for the benzyl viologen-mediated reaction. Purification studies utilizing most of the common methods of protein fractionation were unsuccessful in obtaining a purified preparation of nitrite reductase. The studies implied that the reductase activity is expressed by a multi-component enzyme complex which is highly oxygen labile. Results of a comparative study of several other clostridia indicate that nitrite reductase activities are present only in those clostridial strains which also contain an active hydrogenase and nitrogenase system.
The study of the active center of chymopapain
(University of Hawaii at Manoa, 1966) Tsunoda, Joyce Nishimura
The proteolytic enzyme chymopapain was first isolated from fresh papaya latex by Jansen and Balls (J. Biol. Chem., 137, 459, 1951). Subsequently, a modified procedure for purification of chymopapain from dried papaya latex was developed by Ebata (J. Biol. Chem., 237, 1086, 1962). Chymopapain was shown to be a sulfhydryl enzyme on the basis of its susceptibility to sulfhydryl reagents and its requirement for "thiol activators" such as cysteine and cyanide for maximum activity. Titration of cyanide activated chymopapain with p-chloro mercuribenzoate (CMS) revealed the presence of 1.4-1.6 moles of SH per mole of enzyme (m.w., 30,000). The controlled addition of the colored sulfhydryl reagent, N-4-dimethylamino- 3,5-dinitropheriyl)-maleimide (Witter and Tuppy, Biochim. Biophys. Acta, 45, 429, 1960), and enzymic hydrolysis of the S-DDPS-chymopapain thus formed led to the isolation of a deca-peptide containing the labeled half cysteine residue. The amino acid sequence of this peptide was found to be: Lys-Arg-Val-Pro-Asp-Ser-Gly-Glu-Cys-Tyr (Cys = the labeled half of cysteine residue). This sequence differed from those of the peptides containing the reactive SH groups of papain (Light et al., Proc. Nat'l. Acad. Sci, U.S.A., 52, 1276, 1964) and ficin (Wong and Liener, Biochem. Res. Comm., 11, 470, 1964). v Possible reasons for this difference in the sulfhydryl peptides from papain and ficin and that from chymopapain are discussed. Preliminary studies (Ebata et al., Biochem. Biophys. Res. Comm., 9, 173, 1962) indicated that chymopapain was inhibited by diisopropylfluorophosphate (DFP) under certain conditions. The present investigation showed that there was a corresponding loss of the CMB-titratable SH group and enzymic activity with increasing concentration of DFP added. This corroborated the work of Gould and Liener (Biochemistry, 1, 349, 1965) who reported that an impurity in the DFP preparation reacted with the essential SH group of ficin thus inactivating the enzyme. However, cyanide-activated chymopapain was phosphorylated by DFP (organic phosphorus/protein, 0,89, mole/mole). Unactivated chymopapain was not phosphorylated under the same conditions. The site of phosphorylation was the hydroxyl group of a serine residue. The partial structure of the phosphorylated peptide from tryptic digest of DIP-chymopapain was Ser-Gly(Cys,Asp,Thr,Ser,Glu,Ala)-Lys Methylene-blue catalyzed photooxidation of CMB-chymopapain resulted in the loss of all three histidine residues of chymopapain with only 50% loss of enzymic activity. A tentative mechanism for the reaction between chymopapain and substrate is proposed on the basis of the available data.

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