Method to prepare a neurotrophic composition

Claim 3

The method of claim 1 which further comprises isolating a neurotrophic factor from the protein extract having said trophic effects, which factor has a molecular weight in the range of about 20,000 daltons to about 22,000 daltons as determined by SDS-PAGE analysis.

Claim 4

The method of claim 2 wherein said normal mammal is a rat.

Claim 5

The method of claim 2 wherein said protein fraction is isoelectric at a pH of 5±0.5.

Claim 6

The method of claim 2 wherein said neurotrophic factor has the internal peptide sequence: F-V-Y-A-T-C-N-F-T-L-L-E-L-N-N-A.

Claim 7

The method of claim 1 wherein said extraction comprises homogenizing the skeletal muscle in a buffered aqueous solution comprising one or more protease inhibitors.

Claim 8

The method of claim 6 wherein said aqueous solution comprises one or more chelating agents.

Claim 9

The method of claim 2 wherein said isolation comprises purifying the protein extract by adjusting the extract to pH 5 and collecting the precipitate.

Claim 10

The method of claim 8 wherein said isolation procedure comprises: (i) purifying the precipitate by gel filtration chromatography; and (ii) recovering an .sup.˜ 28 kD protein component.

Claim 11

The method of claim 8 wherein said isolation procedure further comprises: (i) fractionating the precipitate by hydroxylapatite chromatography; and (ii) eluting the bound protein to recover cholinergic factor activity.

Claim 12

The method of claim 8 wherein said isolation procedure further comprises: (i) separating the precipitate by anion exchange chromatography; and (ii) pooling the cholinergically active fractions to recover cholinergic factor activity.

Claim 13

The method of claim 9 wherein said purification procedure further comprises the steps of: (i) fractionating the .sup.˜ 28 kD protein component by hydroxylapatite chromatography; (ii) eluting the bound protein; (iii) separating the eluted protein by anion exchange chromatography; (iv) pooling the cholinergically active fractions; (v) fractionating the pooled activity by hydroxylapatite chromatography; (vi) recovering cholinergic factor activity; and (vii) concentrating the cholinergic factor activity using ultrafiltration.

Claim 14

The method of claim 12 wherein about 0.01% to about 0.1% polyethylene glycol is present in each chromatography step.

Claim 15

The method of claim 12 wherein said purification procedure further comprises subjecting the concentrated cholinergic factor activity to SDS-PAGE analysis and recovering a .sup.˜ 20-22 kD neurotrophic factor.

Claim 16

The method of claim 1 which further comprises isolating a neurotrophic factor from the protein extract having said trophic effects which factor has a molecular weight in the range of about 16,000 to about 18,000 daltons.

Claim 17

The method of claim 15 wherein said normal mammal is a human.

Claim 18

The method of claim 16 wherein said isolation procedure comprises adjusting the pH to 3.5 and adding ammonium sulfate to 100% saturation and collecting the resultant precipitate.

Claim 19

The method of claim 17 wherein said purification procedure further comprises: (i) fractionating the resultant precipitate by ion-exchange chromatography on phosphocellulose; and (ii) recovering bound protein with cholinergic factor activity.

Claim 20

The method of claim 17 wherein said purification procedure further comprises: (i) fractionating the resultant precipitate by gel filtration chromatography; and (ii) pooling the cholinergically active fractions corresponding to an .sup.˜ 17 kD factor to recover cholinergic factor activity.

Claim 21

The method of claim 17 wherein said purification procedure further comprises: (i) subjecting the resultant precipitate to heparin-affinity chromatography; and (ii) recovering cholinergic factor activity

Claim 22

The method of claim 18 wherein said purification procedure further comprises: (i) fractionating the recovered protein by gel filtration chromatography; (ii) pooling the cholinergically active fractions corresponding to an .sup.˜ 17 kD factor; (iii) subjecting the pooled fractions to heparin-affinity chromatography; and (iv) recovering an .sup.˜ 17 kD protein composition.

Claim 23

A protein prepared by the method of claim 2.

Claim 24

A substantially pure neurotrophic factor having a molecular weight of ˜20,000 daltons and an soelectric point of 5±0.5, which contains an internal peptide sequence F-V-Y-A-T-C-N-F-T-L-L-E-L-N-N-A, and which is found in skeletal muscle.

Claim 25

A substantially pure neurotrophic factor having a molecular weight of ˜20,000 daltons and an soelectric point of 5±0.5, which contains an internal peptide sequence F-V-Y-A-T-C-N-F-T-L-L-E-L-N-N-A, and which is found in skeletal muscle.

Claim 26

The neurotrophic factor of claim 23 wherein a K residue precedes the first designated F residue.

Claim 27

A substantially pure neurotrophic factor having a molecular weight of between ˜20,000 and ˜22,000 daltons as measured by SDS FAGE, an isoelectric point of 5±0.5, which neurotrophic factor does not bind substantially to heparin, is substantially non-cross reactive with antibodies to basic PGF, and is found in skeletal muscle.

Claim 28

A substantially pure neurotrophic factor having a molecular weight of between ˜20,000 and ˜22,000 daltons as measured by SDS FAGE, an isoelectric point of 5±0.5, which neurotrophic factor does not bind substantially to heparin, is substantially non-cross reactive with antibodies to basic PGF, and is found in skeletal muscle.

Claim 29

The substantially pure neurotrophic factor of claim 23 which is found in rat muscle tissue.

Claim 30

The substantially pure neurotrophic factor of claim 25 which is found in rat muscle tissue.

Claim 31

A substantailly pure neurotrophic factor having a molecular weight of between ˜20,000 and ˜22,000 daltons as measured by SDS PAGE, and an isoelectric point of 5±0.5, which neurotrophic factor does not bind substantially to heparin, is substantially non-cross reactive with antibodies to basic FGF, comprises the partial amino acid composition as follows: ______________________________________ Residues/mole Amino Acid (mean ± range) ______________________________________ Aspartic Acid 19 ± 1.0 Threonine 8 ± 0 Serine 14 ± 0.5 Glutamic Acid 28 ± 1.5 Proline 8 ± 0.5 Glycine 26 ± 1.0 Alanine 13 ± 0 Valine 11 ± 0 Isoleucine 7 ± 0 Leucine 14 ± 0.5 Tyrosine 6 ± 0.5 Phenylalanine 8 ± 0.5 Histidine 10 ± 0.5 Lysine 12 ± 0.5 Arginine 8 ± 0.5 ______________________________________ and which is found in skeletal muscle.

Claim 32

A substantailly pure neurotrophic factor having a molecular weight of between ˜20,000 and ˜22,000 daltons as measured by SDS PAGE, and an isoelectric point of 5±0.5, which neurotrophic factor does not bind substantially to heparin, is substantially non-cross reactive with antibodies to basic FGF, comprises the partial amino acid composition as follows: ______________________________________ Residues/mole Amino Acid (mean ± range) ______________________________________ Aspartic Acid 19 ± 1.0 Threonine 8 ± 0 Serine 14 ± 0.5 Glutamic Acid 28 ± 1.5 Proline 8 ± 0.5 Glycine 26 ± 1.0 Alanine 13 ± 0 Valine 11 ± 0 Isoleucine 7 ± 0 Leucine 14 ± 0.5 Tyrosine 6 ± 0.5 Phenylalanine 8 ± 0.5 Histidine 10 ± 0.5 Lysine 12 ± 0.5 Arginine 8 ± 0.5 ______________________________________ and which is found in skeletal muscle.

Claim 33

The substantially pure neurotrophic factor of claim 28 which is found in rat muscle tissue.