A phage antibody to the active site of human placental alkaline phosphatase with higher affinity to the enzyme–substrate complex

2007 ◽  
Vol 44 (4) ◽  
pp. 369-376 ◽  
Author(s):  
Vishal Jain ◽  
Deepti Saini ◽  
Pooja Goswami ◽  
Subrata Sinha
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Placental Alkaline Phosphatase ◽  
Phage Antibody ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Human Placental Alkaline Phosphatase

scholarly journals The Promoter of the Rat Gonadotropin-Releasing Hormone Receptor Gene Directs the Expression of the Human Placental Alkaline Phosphatase Reporter Gene in Gonadotrope Cells in the Anterior Pituitary Gland as well as in Multiple Extrapituitary Tissues

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 983-993 ◽  
Author(s):  
Anne Granger ◽  
Valérie Ngô-Muller ◽  
Christian Bleux ◽  
Céline Guigon ◽  
Hanna Pincas ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Reporter Gene ◽  
Transgene Expression ◽  
Pituitary Cells ◽  
Placental Alkaline Phosphatase ◽  
R Gene ◽  
Glycoprotein Hormone ◽  
Steroidogenic Factor 1 ◽  
Functional Sites ◽  
Human Placental Alkaline Phosphatase

Abstract Previous studies dealing with the mechanisms underlying the tissue-specific and regulated expression of the GnRH receptor (GnRH-R) gene led us to define several cis-acting regulatory sequences in the rat GnRH-R gene promoter. These include functional sites for steroidogenic factor 1, activator protein 1, and motifs related to GATA and LIM homeodomain response elements as demonstrated primarily in transient transfection assays in mouse gonadotrope-derived cell lines. To understand these mechanisms in more depth, we generated transgenic mice bearing the 3.3-kb rat GnRH-R promoter linked to the human placental alkaline phosphatase reporter gene. Here we show that the rat GnRH-R promoter drives the expression of the reporter gene in pituitary cells expressing the LHβ and/or FSHβ subunit but not in TSHβ- or GH-positive cells. Furthermore, the spatial and temporal pattern of the transgene expression during the development of the pituitary was compatible with that characterizing the emergence of the gonadotrope lineage. In particular, transgene expression is colocalized with the expression of the glycoprotein hormone α-subunit at embryonic day 13.5 and with that of steroidogenic factor 1 at later stages of pituitary development. Transgene expression was also found in specific brain areas, such as the lateral septum and the hippocampus. A single promoter is thus capable of directing transcription in highly diverse tissues, raising the question of the different combinations of transcription factors that lead to such a multiple, but nevertheless cell-specific, expressions of the GnRH-R gene.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

scholarly journals GnRH Receptor Gene Expression in the Developing Rat Hippocampus: Transcriptional Regulation and Potential Roles in Neuronal Plasticity

Endocrinology ◽  
2010 ◽  
Vol 152 (2) ◽  
pp. 568-580 ◽  
Author(s):  
Anne-Laure Schang ◽  
Valérie Ngô-Muller ◽  
Christian Bleux ◽  
Anne Granger ◽  
Marie-Claude Chenut ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Neuronal Plasticity ◽  
Gnrh Receptor ◽  
Lateral Septum ◽  
Rat Hippocampus ◽  
Marker Genes ◽  
Mrna Levels ◽  
Primary Role ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase

Abstract In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

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