FUNCTIONAL GROUPS IN RHODOPSIN AND THE ROD .into the nervus opticus. Most vertebrates possess two

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FUNCTIONAL GROUPS IN RHODOPSIN AND

THE ROD PHOTORECEPTOR MEMBRANE

W. J. DE GRIP

FUNCTIONAL GROUPS

IN RHODOPSIN ANO

THE ROD PHOTORECEPTOR MEMBRANE

Promotorr : Prof.Dr. S.L. Bonting

Co-Referent : Dr. P.J.M. Daeaien

These investigations were carried out in the depart-

ment of Biochemistry, University of Nijmegen, The Nether-

lands. Additional financial support was received from the

Netherlands Organization for the Advancement of Basic

Research (Z.W.O.), through the Foundation for Chemical

Research in the Netherlands (S.O.N.)

FUNCTIONAL GROUPS IN RHODOPSIN AND

THE ROD PHOTORECEPTOR MEMBRANE

PROEFSCHRIFT

TER VERKRIJGING VAN GRAAD VAN DOCTOR IN DE

WISKUNDE EN NATUURWETENSCHAPPEN

AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN, OP GEZAG VAN

DE RECTOR MAGNIFICUS PROF.MR. F.J.F.M. DUYNSTEE

VOLGENS HET BESLUIT VAN HET COLLEGE VAN DECANEN

IN HET OPENBAAR TE VERDEDIGEN

OP DONDERDAG 28 FEBRUARI 1974

DES NAMIDDAGS TE 4 UUR

DOOR

WILLEM JOHAN DE GRIP

GEBOREN TE APELDOORN

druk : Kripa Repro

Aan Riky

Aan het tot atand komen van dit proefschrift

hebben velen meegewerkt,ieder van wie ik bij deze gaarne

wil bedanken,

In eerate instantie gaat mijn dank uit naar

mevr. A.Valenteijn-Temmink, de heer G.van Gogh en,in

het bizonder, de heer G.L.M.van de Laar voor hun tech-

nische assistentie, en tevens naar mej. B.Renckens voor

haar bijdrage aan de jodaat experimenten (sectie 5.3.3.).

De aminozuur analyses werden uitstekend ver-

zorgd door mej. M.Versteeg, en de heer G.Groenewoud en,

met name, de heer M.G.J.Buys.

Ik ben voorts erkentelijk voor de plezierige

samenwerking met de medewerkers van de afdeling Submi-

croscopische Morfologie, de Instrumentele Dienst en de

afdelingen Medische Illustratie en Fotografie.

Tenslotte dank ik mevr. J.Jansen-Ctoosterman

en met name mevr. C.Hafkenscheid-Albers voor hun

bijdrage aan het typewerk.

ABBREVIATIONS 12

STRUCTURAL ANO FUNCTIONAL ORGANIZATION OF THE ROD 13

1.1. General aspects 13

1.2. The rod photoreceptor membrane 16

1. Isolation 16

2. Composition 17

3. Structure 19

4. Biosynthesis 24

1.3. The rod visual pigment rhodopsin 25

1. Extraction and absorption spectrum 25

2. Purification 28

3. Chromophoric group 30

4. Photolysis 33

5. Regeneration 36

1.4. Excitation and adaptation 38

1. Introduction 38

2. Current status 39

1.5. Aims of this investigation 43

ISOLATION OF BOVINE PHOTORECEPTOR MEMBRANES WITH

MAXIMAL RHODOPSIN CONTENT 46

2.1. Introduction 46

2.2. Materials and methods 48

1. Preparation of 11-cis retinaldehyde 48

2. Isolation of bovine rod outer segment

membranes 49

3. Analytical methods 52

2.3. Results 55

1. Comparison of isolation techniques 55

2. Justification of the isolation procedure 56

3. Purity of the photoreceptor membrane

preparations 63

2.4. Discussion 64

CHEMICAL CHARACTERIZATION OP BOVINE ROO PHOTORECEPTOR MEMBRANES 70 3.1. Introduction 70 3.2. Materials and methods 71

1. Materials 71 2. Protein analysis 71

3. Sugar determinations 74 4. Lipid extraction and analysis 76 5. Determination of primary amino groups 78

3.3. Results 81 1. Chemical composition of lyophilized rod

photoreceptor membranes 81 2. Primary amino groups 84 3. Sulfhydryl groups 93 4. Rhodopsin as photoreceptor membrane protein 94

3.4. Discussion 96

FUNCTIONAL ANALYSIS OP AMINO GROUPS IN THE ROD PHOTORECEPTOR MEMBRANE 100 4.1. Introduction 100 4.2. Materials and methods 104

1. Materials 104 2. Amidination 105 3. Trinitrophenylation 106

4. Modification with fluorobenzene derivatives 106

5. Succinylation 107 6. Analytical techniques 107 7. Assay of retinol:NADP oxidoreductase

activity 107 8. Photolysis 108

4.3. Results 109 1. Amidination 109 2. Trinitrophenylation 118

3. Other reagents 120 4. Photolysis 121

4.4. Discussion 124

1. Chromophoric binding site 124

2. Migration of the chromophore following

illumination 125

3. Retinol:NADP oxidoreductaae 129

4. Structural aspects 131

SULFHYDRYL GROUPS IN THE ROD PHOTORECEPTOR

MEMBRANE 134

5.1. Introduction 134

5.2. Materials and methods 136

1. Materials 136

2. Assay of sulfhydryl groups 136

3. Modification of sulfhydryl groups 136

4. Determination of mercury 138

5. Treatment with iodate 138

5.3. Results 139

1. Exposed sulfhydryl groups 139

2. Modification of sulfhydryl groups 139

3. Iodate effects 145

5.4. Discussion 146

1. Sulfhydryl groups and photolysis of

rhodopsin I46

2. Modification with p-chloromercuribenzoate I48

3. Iodate effect 149

4. Conclusion I52

GENERAL DISCUSSION 154

6.1. Introduction 154

6.2. Action of detergents on the rod photoreceptor

membrane 155

1. Accessibility of amino and sulfhydryl

groups 155

2. Light-induced detergent effects 155

3. Other detergent effects 156

4. Mechanism of action of detergents 157

5. Concluaion 161

6.3. Structure-function relationships in the rod

photoreceptor 161

1. Introduction 161

2. Chromophoric site 162

3. Binding site of retinaldehyde in

photolytic intermediates 163

4. Recombination 163

5. Enzymatic activities 164

6. Structural aspects of the rod

photoreceptor membrane 164

7. Perspective 165

SUMMARY 167

SAMENVATTING 172

REFERENCES 178

CURRICULUM VITAE 198

ABBREVIATIONS

A absorbance

ATP adenoaine 5,-triphosphate

BDH British Drug Houses Ltd, Poole, England

cetyltrimethylammonium bromide

dodecyldimethylaminoxyde (Ammonyx LO)

dinitrofluorobenzene

dodecyltrimethylammonium bromide

dithioerythritol

BTNB 5,5l-dithiobis(2-nitrobenzoic acid) (Ellman's reagent)

molar absorbance coefficient

hr hour

wavelength

MW molecular weight

NADP nicotinamide-adenine dinucleotide phosphate

PCMB p-chloromercuribenzoic acid

PCMBS p-chloromercuribenzene sulphonic acid

SBS sodium dodecylsulfate (sodium laurylsulfate)

TNBS 2,4,6,trinitrobenzene-1-sulphonic acid

TRIS tris(hydroxymethyl)aminomethane

12

CHAPTER 1

STRUCTURAL ANO FUNCTIONAL ORGANIZATION THE ROD

1.1. General aspects

Light perception in the vertebrates is mediated by

the retina, a thin (0.1-0.5 mm) film of tissue, lining

the posterior half of the eye-cup. It consists of three

layers, containing five different cell types, four of

which are nerve cells involved in the processing of the

electrical impulse, generated upon light absorption by

the fifth type, the photoreceptor cells. The latter lie

at the back aide of the retina, bordering the pigment

epithelium. The front side of the retina, bordering the

vitreous body, is covered by nerve fibers concentrating

into the nervus opticus.

Most vertebrates possess two types of photoreceptor

cells, rods and cones, of which the former generally

greatly outnumber the latter. In their functioning as

light receptors the rods and cones are complementary to

each other. The rod has a very high light sensitivity and

functions primarily in twilight (scotopic vision). It can

already be excited by absorption of a single photon

(Hecht et al, 1942; Bouman and van der Velden, 1947). In

further contrast to the cone, which functions under day

light conditions (photopic vision) and is able to discri

minate between light of different wavelengths (color vi

sion), the rod only "perceives" intensity differences

(black-white vision).

Viewed ander an electronmicroscope, the rod cell ap

pears as an elongated tubular structure (Pig. 1), compo

sed of two distinct parts, called the inner segment and

the outer segment, which are connected by a non-motile

cilium.

13

The inner segment contains the common cell organelles

like nucleus, Golgi apparatus, endoplasmatic reticulum and

mitochondria. At the base of the rod, gap junctions may

be formed with neighbouring photoreceptor cells (Raviola

and Gilula, 1973) and synaptic connections of the inva-

ginating type are made with terminals of a horizontal and

a bipolar cell, the first two in the series of nerve cells

(Bowling, 1970; Lasansky, 1972; Boycott and Kolb, 1973a,b),

In contrast, the outer segment has a very unusual

appearance. It contains a pile of hundreds of flat sacs

or discs, which is enclosed by the cell membrane (Pig. 1).

The sacs are formed by invagination of the cell membrane

near the base of the outer segment and are subsequently

pinched off. The sac membranes or photoreceptor membranes

must be the main site of light absorption, since they con-