Post-Translational Processing A Practical Approach

by Higgins, S. J.; Hames, B. D.

• ISBN13: 9780199637959
• ISBN10: 0199637954
• Format: Paperback
• Copyright: 1999-07-29
• Publisher: Oxford University Press

Summary

Post-translational Modification: A Practical Approach is a comprehensive volume of the best current methodology. It is designed to be used at the bench or away from the bench to gain insight into future experimental approaches and (along with its companion volume Protein Expression: A Practical Approach ) forms the final part of the PAS mini-series on protein synthesis and processing. This volume alone covers areas such as protein sequencing, protein folding and import, organelles, and the three major forms of covalent modification -- phosphorylation, glycosylation, and lipid modification. It also addresses proteolytic processing and protein turnover in mammalian cells and yeast. Its extensive breadth of coverage makes it ideal for any biomedical researcher or scientist wanting to investigate protein expression in cell free systems, viruses, and prokaryotic and eukaryotic cells.

Table of Contents

List of Contributors	xv
Abbreviations	xvii
Sequence analysis of expressed proteins	1	(42)
Jeff N. Keen
Alison E. Ashcroft
Introduction	1	(2)
N-terminal sequence analysis	3	(6)
Automated sequencing	3	(6)
Sample preparation	9	(5)
Sample preparation by SDS-PAGE	10	(2)
Sample preparation by HPLC	12	(1)
Other procedures for sample preparation	12	(2)
N-terminal blocking	14	(7)
Protein fragmentation	17	(4)
C-terminal sequencing	21	(4)
Chemical analysis of the C-terminus	21	(1)
Enzymic analysis of the C-terminus	22	(3)
Mass spectrometric analysis of proteins	25	(18)
A brief guide to mass spectrometers	25	(4)
Molecular mass determination	29	(5)
Sequencing by mass spectrometry	34	(6)
References	40	(3)
Protein folding and import into organelles	43	(52)
Joachim Rassow
Introduction	43	(1)
Preparation of preprotein substrates for in vitro import into organelles	44	(5)
Preparation of mRNA for in vitro translation	44	(1)
Synthesis of preproteins in the reticulocyte lysate	44	(2)
Synthesis of preproteins in wheat germ lysate	46	(1)
Synthesis of preproteins in yeast cytosol	47	(1)
Synthesis of preproteins in Escherichia coli	47	(1)
Dihydrofolate reductase as a model protein for import studies	47	(2)
Import of proteins into mitochondria	49	(21)
Isolation of mitochondria for import studies	50	(6)
Import of preproteins into isolated mitochondria	56	(7)
Generation of translocation intermediates	63	(5)
Localization of imported proteins	68	(2)
Import of proteins into microsomes (endoplasmic reticulum)	70	(4)
Isolation of microsomes	70	(3)
Import of preproteins into microsomes	73	(1)
Import of proteins into other organelles	74	(1)
Analysis of protein import into organelles	75	(20)
Monitoring the association of proteins with membranes	75	(2)
Analysis of protein complexes involved in organelle import	77	(13)
Analysis of protein folding after import into organelles	90	(1)
Acknowledgements	91	(1)
References	92	(3)
Analysis of protein phosphorylation	95	(40)
S. Ivar Walaas
Anne Carine Ostvold
Introduction	95	(1)
Investigating protein phosphorylation systems	96	(1)
Phosphorylation of proteins in intact preparations	96	(3)
General considerations	96	(1)
Intact animals	96	(1)
Tissue slices	97	(1)
Isolated cells	98	(1)
Phosphorylation of proteins in cell-free preparations	99	(2)
General considerations	99	(1)
Labelling and stimulation of cell-free preparations	100	(1)
Analysis of phosphorylated proteins	101	(9)
General considerations	101	(1)
Quantification of phosphoproteins	101	(1)
Phosphoprotein separation	102	(1)
Protein isolation by immunomethods	103	(1)
Analysis of multisite phosphorylation	103	(4)
Phosphoamino acid analysis	107	(1)
Analysis of the state of phosphorylation	108	(2)
Analysis of protein kinases	110	(13)
General considerations	110	(1)
Analysis of protein kinase activity in vitro	110	(3)
Analysis of specific protein kinases in vitro	113	(9)
Analysis of protein kinase activity in intact cells	122	(1)
Purification of protein kinases	123	(1)
Phosphoprotein phosphatases	123	(12)
General considerations	123	(1)
P-Ser/P-Thr phosphoprotein phosphatases	123	(4)
Tyrosine-specific phosphoprotein phosphatases	127	(1)
References	128	(7)
Protein glycosylation	135	(40)
David A. Ashford
Fran Platt
Introduction	135	(5)
Protein glycosylation	135	(1)
Oligosaccharide structures	136	(1)
Glycosylation pathways	137	(3)
Characterization of protein glycosylation	140	(1)
Is my protein glycosylated?	140	(4)
Colorimetric methods	140	(1)
Properietary detection methods	141	(1)
Lectin binding	142	(2)
Study of whole protein glycosylation	144	(8)
Detection of N-glycosylation	144	(2)
Monosaccharide composition	146	(3)
Lectin binding analysis	149	(1)
Susceptibility to endoglycosidase H	150	(1)
Other methods	151	(1)
Analysis of glycosylation sites	152	(3)
Protease mapping of glycopeptides	152	(2)
Glycopeptide identification and analysis	154	(1)
Analysis of glycan structure	155	(15)
Glycan release	155	(4)
Glycan labelling	159	(2)
Glycan separation	161	(6)
Structural analysis of glycans	167	(3)
Manipulation of protein glycosylation	170	(5)
References	172	(3)
Lipid modification of proteins	175	(30)
Nigel M. Hooper
R. A. Jeffrey McIlhinney
Introduction	175	(1)
Protein acylation	175	(2)
General consideration	175	(1)
Enzymology	176	(1)
Identification of acylated proteins	177	(11)
General points	177	(2)
Labelling cultured cells with fatty acids	179	(1)
Analysis of acylated proteins	180	(4)
Myristoylated proteins	184	(4)
Glycosyl-phosphatidylinositol membrane anchorage of proteins	188	(2)
Structure of GPI anchors	188	(1)
Signals for attachment of a GPI anchor to a protein	188	(2)
Identification of GPI anchorage	190	(10)
General points	190	(1)
Release of GPI-anchored proteins by bacterial phosphatidylinositol-specfic phospholipase C	191	(3)
Differential detergent solubilization	194	(1)
Detection of the cross-reacting determinant	195	(5)
Metabolic labelling	200	(1)
Prenylation	200	(5)
References	201	(4)
Proteolytic processing	205	(20)
John M. W. Creemers
Elaine M. Bailyes
Iris Lindberg
John C. Hutton
Introduction	205	(3)
Immunoadsorbent assay of PC1 and PC2	208	(5)
Calcium-dependence of prohormone convertases	212	(1)
Expression of prohormone convertases in DG44 CHO cells	213	(3)
Fluorogenic assay for PC1 and PC2	216	(4)
PC1 assay	217	(1)
PC2 assay	218	(2)
Transient expression with recombinant vaccinia virus V.V.:T7	220	(5)
Introduction	220	(2)
References	222	(3)
Protein degradation in mammalian cells	225	(40)
Aaron Ciechanover
Bernd Wiederanders
The ubiquitin-proteasome pathway in mammalian cells	225	(19)
Introduction	225	(4)
Preparation of cell extracts for monitoring conjugation and degradation	229	(1)
Fractionation of cell extracts for monitoring conjugation and degradation	230	(3)
Labelling of proteolytic substrates	233	(3)
Conjugation of proteolytic substrates	236	(5)
Degradation of proteolytic substrates	241	(1)
Use of inhibitors to study proteasome function	241	(3)
Proteolysis in mammalian lysosomes	244	(21)
Introduction	244	(1)
Isolation of mammalian lysosomes	245	(6)
Lysosomal peptidases	251	(6)
Measurement of lysosomal protein degradation	257	(4)
Acknowledgementsx	261	(1)
References	261	(4)
Protein degradation and proteinases in yeast	265	(38)
Wolfgang Hilt
Dieter H. Wolf
Introduction	265	(3)
Advantages of yeast for studying eukaryotic cell biology	266	(2)
Growth of yeast cells and preparation of cell extracts	268	(2)
Analysis of protein turnover	270	(5)
Protein degradation in vivo	270	(2)
Degradation of individual proteins	272	(3)
The proteasome and protein degradation in yeast	275	(8)
Proteinase yscD, a major cytoplasmic peptidase	283	(2)
Introduction	283	(1)
Assay of proteinase yscD	284	(1)
Protein degradation in the vacuole	285	(18)
Introduction	285	(1)
Assay of vacuolar peptidases	286	(8)
Purification of yeast vacuolar proteinases	294	(5)
Isolation of yeast vacuoles	299	(1)
Acknowledgements	300	(1)
References	301	(2)
Appendix	303	(6)
Index	309