| 1. |
- Ek, P, et al.
(författare)
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Exogenous protein kinases A and C, but not endogenous prostasome-associated protein kinase, phosphorylate semenogelins I and II from human semen
- 2002
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Ingår i: Journal of Andrology. - 0196-3635. ; 23:6, s. 806-814
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Tidskriftsartikel (refereegranskat)abstract
- Semenogelins I and II are the quantitatively dominating proteins in human semen. They comprise the major part of the sperm-entrapping gel formed at ejaculation, which subsequently liquefies due to proteolysis of the gel-forming proteins by prostate-specific antigen (PSA). The mechanism behind gel formation and its physiological significance is not known. We have studied phosphorylation and dephosphorylation of human semenogelins. Both were phosphorylated by protein kinases A and C (PKA and PKC, respectively) at a rate about 5 times less than that of histone. For PKA, incorporated ( P)phosphate into semenogelin approached a maximum above 1 mol/mol. Corresponding values for phosphorylation of the semenogelins with PKC were greater than 10. There was no change in the sensitivity of phosphosemenogelins to proteolysis by PSA. Serine (PKA) and serine and threonine (PKC) were the phosphate-accepting amino acid residues, and all incorporated (P-32)phosphate could be removed from the semenogelins with human acid phosphatase. Nil or very little phosphate could be detected in purified semenogelins isolated from seminal plasma. In vivo, about half the protein kinase activity in seminal plasma was bound to prostasomes. PKA but not PKC purified from prostasomes could phosphorylate specific substrates, but they could phosphorylate either of the semenogelins.
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| 2. |
- Ressine, Anton, et al.
(författare)
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Macro/Nano-Structured Silicon as Solid Support for Antibody Arrays: Surface Design, Reproducibility, and Assay Characteristics Enabling Discovery of Kallikrein Gene Products for Prostate Disease Diagnostics
- 2005
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Ingår i: Nanobiotechnology. - 1551-1286. ; 1:1, s. 93-104
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Tidskriftsartikel (refereegranskat)abstract
- To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro- and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 104-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.
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