Problem Vitamin C and collagen synthesis

Clinically, scurvy (section 11.14.3) is characterized by fragility of the blood vessel walls and small subcutaneous haemorrhages around hair follicles (petechial haemorrhages), as well as inflammation of the gums and loss of the dental cement (and hence loss of teeth) and poor healing of wounds. In advanced cases there is intense deep bone pain, and there may be degenerative changes in the heart, leading to cardiac emergency. In some cases there are also mood changes (indeed, scurvy is the old English word for ill-tempered). It was known from the studies of Lind in 1757 that fresh orange and lemon juice would prevent or cure scurvy; studies during the early part of the twentieth century identified the protective or curative factor as ascorbic acid or vitamin C (see Figure 11.28).

Studies in the 1940s showed that in vitamin C deficiency there was poor healing of wounds, and the scar tissue was weak with little collagen present. The results of studies of the formation of collagen in granulomatous tissue from guinea pigs at different levels of vitamin C nutrition in response to subcutaneous injection of carrageenan are shown in Table 9.14.

What conclusions can you draw from these results?

Table 9.14 Percentage composition of granulatomous tissue in vitamin C-deficient guinea pigs after subcutaneous injection of carageenan

Control

Vitamin C-

animals

deficient animals

Collagen

11.6 ± 0.64

2.3 ± 0.14

Water

66.0

85.0

From data reported by Robertson WvB and Schwartz B (1953) Journal of Biological Chemistry 201: 689— 696.

From data reported by Robertson WvB and Schwartz B (1953) Journal of Biological Chemistry 201: 689— 696.

Isolated fibroblasts synthesize and secrete collagen. If they are incubated in the presence of low vitamin C, they secrete only a very small amount of collagen, and a soluble, gelatine-like protein accumulates in the rough endoplasmic reticulum. Similarly, incubation of fibroblasts under anaerobic conditions, or in the presence of iron-chelating compounds, also prevents the secretion of normal collagen and the accumulation of the soluble gelatine-like protein in the rough endoplasmic reticulum.

What conclusions can you draw from these observations?

One of the characteristic features of collagen is its relatively high content of hydroxyproline (Hyp). Table 9.15 shows the incorporation of radioactivity into guinea pig granuloma tissue incubated with [14C]proline or [14C]hydroxyproline.

What conclusions can you draw from these results?

Figure 9.14 shows the incorporation of [14C]proline into proline and hydroxyproline in a cell-free system from chick embryos.

What conclusions can you draw from these results?

Table 9.16 shows the incorporation of [3H]proline into hydroxyproline in collagen formed by granuloma tissue from control and vitamin C-deficient (scorbutic) guinea pigs, and Table 9.17 the effects of various inhibitors of transcription and translation on the stimulation by ascorbate of proline hydroxylation in mouse fibroblasts in culture.

What conclusions can you draw from these results?

Prolyl hydroxylase has been purified. It requires ascorbate, molecular oxygen and iron (Fe2+) for activity. There is no change in the redox state of the iron during the reaction. In addition, with the purified enzyme there is an absolute requirement for a-ketoglutarate for activity. Table 9.18 shows the disappearance of ketoglutarate and appearance of hydroxyproline on incubation of purified prolyl hydroxylase.

What is the approximate ratio of ketoglutarate utilised/hydroxyproline formed?

What conclusions can you draw from these results?

Studies with 18O2 showed that one atom of oxygen is incorporated into hydroxyproline and the other into succinate formed by oxidative decarboxylation of ketoglutarate.

What conclusions can you draw from these results?

There is oxidation of ascorbate during the hydroxylation of proline, but very much less than 1 mol of ascorbate is oxidized per mol of hydroxyproline or succinate formed,

Table 9.15 Incorporation of radioactivity into collagen from {14C}proline and {14C}hydroxyproline

Tissue incubated with

Radioactivity (dpm/^mol) in

[14C]Proline

[14C]Hydroxyproline

Free amino acids in tissue

2400

3500

Hydroxyproline in collagen

465

2.3*

In samples maintained at 4 °C there was apparent incorporation of 2—2.5 dpm/|J,mol into hydroxyproline. From data reported by Green NM and Lowther DA (1959) Biochemical Journal 71: 55—66.

In samples maintained at 4 °C there was apparent incorporation of 2—2.5 dpm/|J,mol into hydroxyproline. From data reported by Green NM and Lowther DA (1959) Biochemical Journal 71: 55—66.

9000

6000

3000

6000

3000

_--II

Pro -■-Hyp

50 100

minutes incubated

Figure 9.14 Incorporation of label from {14C}proline into proline and hydroxyproline in proteins synthesized by a cell-free system. From data reported by Peterkovsky B and Udenfriend S (1963) Journal of Biological Chemistry 38: 3966-3977.

Table 9.16 Incorporation of radioactivity from {3H}proline into hydroxyproline in collagen formed by granuloma tissue from control and vitamin C-deficient (scorbutic) guinea pigs

Incubation time (min)

Radioactivity (dpm) in hydroxyproline in tissue from

Control animals

Vitamin C-deficient animals

30 60 120

2400 5070 8700

100 150 360

From data reported by Stone N and Meister A (1962) Nature 194: 555-557.

Table 9.17 The effects of various inhibitors of transcription and translation on the stimulation by ascorbate of proline hydroxylation in mouse fibroblasts in culture

Hydroxyproline

dpm per mg protein

No addition

27,700

+ 2.5 x I0-4 mol/L ascorbate

58,800

+ Actinomycin D, then ascorbate 15 minutes later

58,700

+ Puromycin, then ascorbate I5 minutes later

62,900

+ Cycloheximide, then ascorbate I5 minutes later

65,100

From data reported by Stassen FLH, Cardinale GJ and Udenfriend S (1973) Proceedings of the National Academy of Sciences of Sciences of the USA 70: 1090.

From data reported by Stassen FLH, Cardinale GJ and Udenfriend S (1973) Proceedings of the National Academy of Sciences of Sciences of the USA 70: 1090.

Table 9.18 The disappearance of a-ketoglutarate and appearance of hydroxyproline during incubation of purified prolyl hydroxylase a-Ketoglutarate (nmol)

Initial Remaining Utilized Proline hydroxylated (nmol)

  1. 1 1.3 ? 13.7
  2. 6 3.2 ? 28.2
  3. 0 8.0 ? 40.1
  4. 6 16 ? 47.7

From data reported by Rhoads RE and Udenfriend S (1968) Proceedings of the National Academy of Sciences of the USA 60: 1473.

or of O consumed. Figure 9.15 shows the hydroxylation of proline-containing peptides by purified prolyl hydroxylase in the presence and absence of ascorbate. The upper graph shows the results for incubation over 1 minute; during the first 10 seconds the enzyme catalyses hydroxylation of ~30 mol of proline per mole of enzyme in the absence of ascorbate. The lower graph shows the results of incubation over 5 minutes,

time (seconds)

Figure 9.15 Activity of purified prolyl hydroxylase incubated with and without ascorbate. From data reported by Myllyla R et al. (1978) Biochemical and Biophysical Research Communications 83: 441-448.

time (minutes)

Figure 9.15 Activity of purified prolyl hydroxylase incubated with and without ascorbate. From data reported by Myllyla R et al. (1978) Biochemical and Biophysical Research Communications 83: 441-448.

as well as the addition of ascorbate to the ascorbate-free incubation after 3 minutes. After 2 minutes' incubation without added ascorbate the protein-bound iron had been oxidized from Fe2+ to Fe3+, and was reduced back to Fe2+ on addition of ascorbate.

What conclusions can you draw from these results? Can you explain the role of ascorbate in proline hydroxylation ?

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