Evidence for histidine as another functional group of δ-aminolevulinic acid dehydratase from beef liver

δ-Aminolevulinic acid dehydratase (EC 4.2.1.24) was obtained in highly purified form from beef liver. Upon photooxidation of the enzyme in the presence of methylene blue as a sensitizer led to a loss of the enzymatic activity according to pseudo-first order kinetics. The pronounced pH dependence (pk value of 6.8) of the photooxidation rate and the results of amino acid analysis suggested that the inactivation was largely due to the modification of the histidine residue. The finding of the enzyme with little activity in the presence of diethylpyrocarbonate was consistent with such a speculation. On the basis of these results, it can be postulated that the histidine residue seems to play an important role in the enzymatic activity of δ-aminolevulinic acid dehydratase.     

Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease

Kinetic constants for the transesterification of eight dinucleoside phosphates CpX and UpX by bovine and turtle pancreatic ribonuclease were determined. Both ribonucleases have a preference for purine nucleotides at the position X. However, bovine ribonuclease, like other mammalian ribonucleases, prefers 6-amino bases at this site, while turtle ribonuclease prefers 6-keto bases. This difference in specificity at the B2 site may be explained by the substitution of glutamic acid at position 111 by valine in turtle ribonuclease. These results have been confirmed by inhibition studies with the four nucleoside triphosphates. Inhibition studies with pT and pTp showed that a cationic binding group (P0) for the 5'-phosphate of the pyrimidine nucleotides bound at the primary B1 site is present in turtle ribonuclease, although lysine at position 66 in bovine ribonuclease is absent in turtle ribonuclease. However, the side chain of lysine 122 in turtle ribonuclease is probably located in the correct position to take over the role as cationic P0 site.     

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.     

In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development.

Previous studies on mice bearing various mutations within the c-kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c-kit gene and a respectable understanding of the molecular nature of c-kit, however, it is not clear at what stage of gestation c-kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti-c-kit antibody, ACK2, as an antagonistic blocker of c-kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid-gestation, c-kit is functionally required during a restricted period around day 14.5 post-coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c-kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.     

Modulation of postinhibitory rebound rise in plasma GH by hypothalamic hormones in patients with acromegaly

To evaluate the GH regulating mechanism in acromegalic patients, post-inhibitory rebound rise in GH secretion induced by somatostatin was studied in these cases and normal subjects, and was compared with the rebound GH rise induced by dopamine. After somatostatin infusion (500 micrograms/75 min) both 5 normal and 9 acromegalic subjects showed prompt GH decreases during the infusion (% decrease: 69.1 +/- 10.4 vs 74.4 +/- 5.1) and showed rebound rises after its termination. However, the rebound rises occurred more promptly and markedly in normal controls than in acromegalic patients, i.e. the rebound peak appeared at 45 min in normal controls and at 75 min in acromegalic patients after the cessation of somatostatin infusion. Dopamine (DA) infusion (5 micrograms/kg/min for 90 min) also induced similar inhibition and postinhibitory rebound rises in GH secretion in 7 patients with acromegaly. Although the maximum inhibition (65.6 +/- 6.4% vs 74.4 +/- 5.1%) and the inhibitory area (4338.0 +/- 481.5% X min vs 3682.5 +/- 295.5% X min) during the DA or somatostatin infusion were not different, the rebound at 15 min was significantly greater after DA than after somatostatin (p less than 0.02). When TRH was injected at the termination of somatostatin infusion, the rebound increase was significantly enhanced and the rebound peak appeared 45 min earlier than after a single somatostatin administration. Similarly, hp GRF (1-44)-NH2 enhanced the postinhibitory rebound rises in 4 patients studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3',5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in Ellsworth-Howard test

The effect of synthetic 1-34 fragment of human parathyroid hormone (hPTH(1-34] on plasma adenosine 3',5'-monophosphate (cAMP) in human subjects and the diagnostic criteria for the plasma cAMP response in an Ellsworth-Howard test were studied. 20 or 30 micrograms hPTH(1-34) and 200 USP Parathormone (Eli Lilly & Co.), infused intravenously over 5 min, produced very similar patterns of response in plasma cAMP, peak values being observed within 5 or 10 min after the end of the infusion. The maximum levels of plasma cAMP were over 111.5 pmol/ml in all of the normal subjects (n = 5) and patients with idiopathic hypoparathyroidism (n = 22), including those of children, but the plasma cAMP did not rise above 65.0 pmol/ml in pseudohypoparathyroidism (n = 7). There existed a significant correlation between the maximum plasma cAMP concentrations and increases in urinary cAMP excretion after infusions of both hPTH(1-34) and Parathormone. These results suggest that hPTH(1-34) has effects essentially identical to those of native PTH on plasma cAMP. We would like to propose a new diagnostic criterion in the Ellsworth-Howard test: a peak value of plasma cAMP over 100 pmol/ml after 30 micrograms hPTH(1-34) infusion is regarded as a normal response.     

Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.