Porcine kidney leucine aminopeptidase ((alpha)-amino-acyl peptide hydrolase, EC 3.4.11.1) has been purified by affinity chromatography over L-leucyglycyl-AH-Sepharose. The purified enzyme contains 1 gram atom per subunit of catalytically essential Zn('2+). Activation by Mg('2+) or Mn('2+), and inhibition by Cu('2+), Ni('2+), Zn('2+) or Hg('2+) is due to the binding of these ions to a second, regulatory site on each subunit. The catalytic pathway for the hydrolysis of peptides by metalloleucine aminopeptidases has been investigated in cryosolvents at subzero temperatures. The enzyme is very stable and is not denatured at 23(DEGREES)C in the presence of 50% v/v methanol, ethanol, dimethylsulfoxide or dimethylformamide. The effect of cosolvent concentration at 23(DEGREES)C is to decrease k(,cat) linearly and increase K(,M) exponentially. Arrhenius plots for peptide hydrolysis in the absence or presence of methanol give activation energies that agree favorably. All evidence shows that methanol does not perturb the catalytic pathway and is, therefore, a suitable cryosolvent for investigations at subzero temperatures. Direct visualization of E(.)S intermediates formed during the hydrolysis of L-Leu-Gly-NHNH-Dns by various {(LAP)Zn(,6)Me(,6)} in 50% v/v methanol, 0.1 M KCl, 1 mM Me('2+), 10 mM Tris, pH* 9.0 at subzero temperature has been carried out using the fluorescence energy transfer technique. Results show that the interconversion of (E(.)S)(,1) and (E(.)S)(,2) is detectable at -30(DEGREES)C or lower. A minimal reaction pathway that includes these two intermediates has been proposed. The regulating metal ion exerts a marked influence on both the steady-state and pre-steady-state kinetic parameters for this pathway. / Source: Dissertation Abstracts International, Volume: 43-09, Section: B, page: 2883. / Thesis (Ph.D.)--The Florida State University, 1982.

The specific collagenase stored in the granules of human neutrophils has been purified by a simple, reproducible method. A sensitive, accurate and convenient assay for tissue collagenases has been developed to aid the purification. This assay, which is based on the hydrolysis of ('3)H acetylated rat tail tendon type I collagen, has also enabled the inhibition of this enzyme to be studied quantitatively. The purified enzyme has very high specific activity towards type I collagen and is devoid of other contaminating proteolytic activities. It is a zinc metalloproteinase that requires calcium ions for activity. In addition, it contains essential lysine, tyrosine and carboxyl residues within the active site. / The types of compounds that have been found to be good inhibitors for other zinc metalloproteinases also inhibit both the human neutrophil and the class I and class II Clostridial collagenases. The Clostridial enzymes are strongly inhibited by phosphonoamidates and ketone analogs that contain collagen-like sequences. Both kinds of compounds are competitive inhibitors with K(,I) values in the micromolar range. The tightest binding inhibitor is CN-Leu('K)-Gly-Pro-Ala, which binds to (epsilon)-collagenase with a K(,I) of 110 nM. Phosphonoamidates are also tight binding inhibitors of human neutrophil collagenase. The best inhibitor has a K(,I) value of 35 (mu)M for this enzyme. / This pattern of inhibition suggests a remarkable similarity between the mechanism of action of these collagenases and other well studied zinc metalloproteinases. The inhibition data also suggest the possible use of these inhibitors as pharmaceuticals and as affinity ligands for the purification of the collagenases. In fact, the use of the ketone analogs to purify the class I and class II Clostridial collagenases from crude mixtures has been successfully demonstrated. / Source: Dissertation Abstracts International, Volume: 47-06, Section: B, page: 2418. / Thesis (Ph.D.)--The Florida State University, 1986.

This is a study of the binding of the thyroid hormone (TH), triiodothyronine (T(,3)), to nuclei of red blood cells (RBCs) of Rana catesbeiana tadpoles. Specific binding activity was detected and the number of T(,3) binding sites per nucleus increased during spontaneous or T(,3)-induced metamorphosis. These results suggested that the binding sites are T(,3) receptors and that the increase in their number is a direct response of the RBCs to T(,3), a receptor induction. The proposal of receptor induction was investigated further. / The increase in the number of T(,3) binding sites after a T(,3) injection was completely inhibited by cycloheximide (CH) or actinomycin-D (act-D), inhibitors of protein synthesis and RNA synthesis, respectively. However, if either inhibitor was administered 48 hours or more after T(,3) injection the increase was not inhibited. The inhibitors were only effective when administered before T(,3) or within 24 hours after T(,3). This requirement for protein synthesis and RNA synthesis is typical of responses to thyroid hormone (TH) in amphibian metamorphosis. / When tadpole RBCs were incubated with T(,3) in vitro in M199, a nutrient rich medium, the number of nuclear T(,3) binding sites was constant during the first 24 hours and then increased over the next 24 hours. Increased T(,3) binding in vitro was highly sensitive to inhibition by CH (10('-6) M) or act-D (0.04 micrograms/ml). These results demonstrate that tadpole RBCs respond directly to T(,3) and that the increased T(,3) binding requires synthesis of RNA and protein in the RBCs. / Stimulation of increased nuclear T(,3) binding in vitro occurred over the same range of T(,3) concentration as occupation of the binding sites. The T(,3) binding sites had a high affinity for TH analogs which are very potent in stimulating metamorphic responses and a low affinity for analogs which are relatively inactive. These properties support the identification of the T(,3) binding sites as TH receptors. / The results of these studies support the concept of TH receptor induction by TH in tadpole RBCs during metamorphosis. / Source: Dissertation Abstracts International, Volume: 47-01, Section: B, page: 0187. / Thesis (Ph.D.)--The Florida State University, 1985.

Source: Dissertation Abstracts International, Volume: 31-09, Section: B, page: 5167. / Thesis (Ph.D.)--The Florida State University, 1970.

Source: Dissertation Abstracts International, Volume: 32-09, Section: B, page: 5077. / Thesis (Ph.D.)--The Florida State University, 1971.

Source: Dissertation Abstracts International, Volume: 30-02, Section: B, page: 0536. / Thesis (Ph.D.)--The Florida State University, 1968.

Source: Dissertation Abstracts International, Volume: 30-06, Section: B, page: 2577. / Thesis (Ph.D.)--The Florida State University, 1969.

The H-2K('b) major histocompatibility complex glycoprotein antigens from EL4 and from a carbohydrate-variant subline, WD1, were purified on an anti-H-2K('b) (B8-24-3) monoclonal antibody affinity column. Previous studies have demonstrated that the cell-surface forms of the H-2 molecules from the WD1 variant line are complex but lack the terminal sialic acid residues. Each purified antigen was reconstituted along with purified cytoskeletal proteins into membrane vesicles of defined lipid composition, protein composition, and lipid/protein ratio. / A sensitive microassay was refined to detect the purified H-2K('b) antigens in the liposomes. The assay utilizes the liposomes to inhibit specific conjugate formation between allogeneically primed (H-2('d)) anti-H-2('b)) cytotoxic T cells and H-2('b) target cells. As little as 5 ng of purified H-2K('b) plus 23 ng cytoskeletal proteins in vesicles inhibited conjugate formation to 50% of the maximum inhibition observed. This inhibition was shown to be specific in two ways: (1) control vesicles containing purified H-2('k) proteins inhibited conjugate formation but only at 5- to 7-fold higher H-2('k) concentrations, and (2) the same H-2K('b)-containing vesicles did not inhibit nonspecific conjugate formation. / Liposomes containing H-2K('b) from either EL4 or WD1 similarly inhibited specific conjugate formation between EL4 target cells and the allogeneically primed cytotoxic T cells. These results suggest that the presence or absence of the terminal sialic acid residues on the two N-asparagine-linked complex carbohydrates of H-2K('b) did not affect the recognition step between the CTL and vesicles. / Source: Dissertation Abstracts International, Volume: 45-09, Section: B, page: 2902. / Thesis (Ph.D.)--The Florida State University, 1984.

The substrate specificity of the collagenases from Clostridium histolyticum has been investigated by measuring the rates of hydrolysis of more than eighty peptides covering the P(,6) to P(,5)' binding sites of the enzymes. The choice of peptides was patterned after sequences found in the (alpha)1 and (alpha)2 chains of Type I collagen. To accomplish this, three continuously recording spectrophotometric assays have been developed. Each peptide contained either a 2-furanacryloyl or cinnamoyl group on position P(,2) or the 4-nitrophenylalanyl residue in position P(,1). Hydrolysis of the P(,1)-P(,1)' bond produces an absorbance change in thesse chromophores that has been used to quantitate the rate of hydrolysis of these peptides under first order conditions ({S}<<K(,m)) from which k(,cat)/K(,m) values have been calculated. The identity of the amino acid in all six positions (P(,3)-P(,3)') or elongation of the peptide to positions P(,6) or P(,5)' markedly influences the hydrolysis rates. This indicates that the enzymes have an extended active site. All six enzymes have a strict requirement for Gly in position P(,1)' and a preference for Gly in position P(,3). In general, class I enzymes (Bond and Van Wart, Biochemistry, 23, 3085, (1984)) prefer substrates with Pro or Ala in position P(,2)' and Hyp, Arg or Ala in position P(,3)'. Class II enzymes show a much broader specificity in positions P(,1)' through P(,3)' and are able to hydrolyze peptides containing all of the common triplets found in collagen. The complementary specificities between the classes of enzymes may explain the observed synergistic digestion of collagen by these enzymes. All six enzymes prefer hydrophobic residues in position P(,1) although the residues commonly found in collagen are hydrolyzed well. The enzymes show a preference for a correctly spaced Gly or Pro residue in the P(,3) to P(,1) / triplet and hydrolyze Gly-Pro, Gly-Ala or Ala-Pro sequences best. These studies indicate that the previously established rules for the specificity of these enzymes require modification. A new collagenase has been isolated (designated -collagenase) and purified to homogeneity. Its substrate specificity clearly indicates it is a class I enzyme. Finally, the mechanism of degradation of extended peptides has been investigated. / Source: Dissertation Abstracts International, Volume: 45-09, Section: B, page: 2907. / Thesis (Ph.D.)--The Florida State University, 1984.

Histones are synthesized coordinately with DNA during the S phase of the cell cycle. However, mouse myeloma cells synchronized by isoleucine starvation synthesize increased amounts of H1 relative to the core histones. The synthesis of H1 increases coordinately again with core histones during S phase. Hydroxyurea blocks the H1 synthesis in isoleucine-starved cells but cytosine arabinoside only partially inhibits this H1 synthesis. When cells were starved for lysine, the relative proportion of the H1 synthesized was much lower than normal. These changes in relative ratios of synthesis are reflected in changes in mRNA levels. Although these phenomena can be explained by a "trivial" translational mechanism, 3T3 cells starved for serum also synthesized proportionally higher amounts of H1 as compared to core histones. The increased H1 synthesis in isoleucine starved cells could be the very reason why isoleucine starvation arrests cultured mammalian cells in G(,1). / Our recent isolation of mouse histone genes (Sittman et al., Proc. Natl. Acad. Sci. 78, 4078 (1981) allows quantitation of the levels of histone mRNA in cultured mammalian cells. I have measured the levels of H3 and H2b mRNA by Northern hybridization and in vitro translation in four cell lines: myeloma cells synchronized by isoleucine starvation, 3T3 cells synchronized by serum starvation, lymphoma S49 cells selected at G(,1) or S phase by centrifugal elutriation, and CHO cells synchronized by mitotic shake-off. The results indicate that histone mRNA levels are regulated during the cell cycle. However, there are significant levels of histone mRNA found in G(,1) in myeloma cells, S49 cells and CHO cells (30-50% of S phase). These RNAs accumulate in G(,1) in myeloma cells during isoleucine starvation. In contrast, 3T3 cells arrested in G(,1) by serum starvation do not contain detectable (< 2%) histone mRNA. One step of the regulation is at the level of RNA turnover. Treatment with hydroxyurea reduced histone mRNA levels by 85% in one hour. These results are consistent with histone mRNA synthesis being initiated in G(,1) and the mRNA being destroyed at the end of S phase. / Source: Dissertation Abstracts International, Volume: 42-10, Section: B, page: 4043. / Thesis (Ph.D.)--The Florida State University, 1981.